Tetracycline-regulated transcriptional inhibitors

ABSTRACT

Nucleic acid molecules and proteins useful for regulating the expression of genes in eukaryotic cells and organisms in a highly controlled manner are disclosed. In the regulatory system of the invention, transcription of a tet operator-linked nucleotide sequence is inhibited by a transcriptional inhibitor fusion protein composed of two polypeptides, a first polypeptide which binds to tet operator sequences either (i) in the absence but not the presence of tetracycline (or an analogue thereof) or (ii) in the presence but not the absence of tetracycline (or an analogue thereof), and a second polypeptide which directly or indirectly inhibits transcription in eukaryotic cells. In one embodiment, the fusion protein comprises a Tet repressor operatively linked to a transcriptional silencer polypeptide. In another embodiment, the fusion protein comprises a mutated Tet repressor operatively linked to a transcriptional silencer polypeptide. The fusion proteins of the invention are useful for reducing the level of transcription of a tet operator-linked target gene. Moreover, the fusion proteins of the invention can be used in combination with tetracycline-regulated transcriptional activator fusion proteins to allow for precise regulation of the expression of one or multiple target genes. Kits including the components of the regulatory system of the invention are also encompassed by the invention.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 08/275,876,filed Jul. 15, 1994, now U.S. Pat. No. 5,654,168, which is acontinuation-in-part of U.S. Ser. No. 08/270,637, filed Jul. 1, 1994,now abandoned, and a continuation-in-part of U.S. Ser. No. 08/260,452,filed Jun. 14, 1994, now U.S. Pat. No. 5,650,298 which is acontinuation-in-part of U.S. Ser. No. 08/076,327, filed Jun. 14, 1993,now abandoned, and a continuation-in-part of U.S. Ser. No. 08/076,726,filed Jun. 14, 1993, now U.S. Pat. No. 5,464,758. The contents of eachof these applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Functional analysis of cellular proteins is greatly facilitated throughchanges in the expression level of the corresponding gene for subsequentanalysis of the accompanying phenotype. For this approach, an inducibleexpression system controlled by an external stimulus is desirable.Ideally such a system would not only mediate an "on/off" status for geneexpression but would also permit limited expression of a gene at adefined level.

Attempts to control gene activity have been made using various inducibleeukaryotic promoters, such as those responsive to heavy metal ions (Mayoet al. (1982) Cell 29:99-108; Brinster et al. (1982) Nature 296:39-42;Searle et al. (1985) Mol. Cell. Biol. 5:1480-1489), heat shock (Nouer etal. (1991) in Heat Shock Response, e.d. Nouer, L. , CRC, Boca Raton,Fla., pp 167-220) or hormones (Lee et al. (1981) Nature 294:228-232;Hynes et al. (1981) Proc. Natl. Acad. Sci. USA 78:2038-2042; Klock etal. (1987) Nature 329:734-736; Israel & Kaufman (1989) Nucl. Acids Res.17:2589-2604). However, these systems have generally suffered from oneor both of the following problems: (1) the inducer (e,g, heavy metalions, heat shock or steroid hormones) evokes pleiotropic effects, whichcan complicate analyses, and (2) many promoter systems exhibit highlevels of basal activity in the non-induced state, which preventsshut-off the regulated gene and results in modest induction factors.

An approach to circumventing these limitations is to introduceregulatory elements from evolutionarily distant species such as E. coliinto higher eukaryotic cells with the anticipation that effectors whichmodulate such regulatory circuits will be inert to eukaryotic cellularphysiology and, consequently, will not elicit pleiotropic effects ineukaryotic cells. For example, the Lac repressor (lacR)/operator/inducersystem of E. coli functions in eukaryotic cells and has been used toregulate gene expression by three different approaches: (1) preventionof transcription initiation by properly placed lac operators at promotersites (Hu & Davidson (1987) Cell 48:555-566; Brown et al. (1987) Cell49:603-612; Figge et al. (1988) Cell 52:713-722; Fuerst et al. (1989)Proc. Natl. Acad. Sci. USA 8:2549-2553: Deuschle et al. (1989) Proc.Natl. Acad. Sci. USA 86:5400-5405); (2) blockage of transcribing RNApolymerase II during elongation by a LacR/operator complex (Deuschle etal. (1990) Science 2:480-483); and (3) activation of a promoterresponsive to a fusion between LacR and the activation domain of herpessimples virus (HSV) virion protein 16 (VP16) (Labow et al. (1990) Mol.Cell. Biol. 10:3343-3356; Baim et al. (1991) Proc. Natl. Acad. Sci. USA88:5072-5076).

In one version of the Lac system, expression of lac operator-linkedsequences is constitutively activated by a LacR-VP16 fusion protein andis turned off in the presence of isopropyl-β-D-thiogalactopyranoside(IPTG) (Labow et al. (1990), cited supra). In another version of thesystem, a lacR-VP16 variant is used which binds to lac operators in thepresence of IPTG, which can be enhanced by increasing the temperature ofthe cells (Baim et al.(1991), cited supra). The utility of these lacsystems in eukaryotic cells is limited, in part, because IPTG actsslowly and inefficiently in eukaryotic cells and must be used atconcentrations which approach cytotoxic levels. Alternatively, use of atemperature shift to induce gene expression is likely to elicitpleiotropic effects in the cells. Thus, there is a need for a moreefficient inducible regulatory system which exhibits rapid and highlevel induction of gene expression and in which the inducer is toleratedby eukaryotic cells without cytotoxicity or pleiotropic effects.

Components of the tetracycline (Tc) resistance system of E. coli havealso been found to function in eukaryotic cells and have been used toregulate gene expression. For example, the Tet repressor (TetR), whichbinds to tet operator sequences in the absence of tetracycline andrepresses gene transcription, has been expressed in plant cells atsufficiently high concentrations to repress transcription from apromoter containing tet operator sequences (Gatz, C. et al. (1992)Plants 2:397-404). However, very high intracellular concentrations ofTetR are necessary to keep gene expression down-regulated in cells,which may not be achievable in many situations, thus leading to"leakiness" in the system.

In other studies, TetR has been fused to the activation domain of VP16to create a tetracycline-controlled transcriptional activator (tTA)(Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA89:5547-5551). The tTA fusion protein is regulated by tetracycline inthe same manner as TetR, i.e., tTA binds to tet operator sequences inthe absence of tetracycline but not in the presence of tetracycline.Thus, in this system, in the continuous presence of Tc, gene expressionis kept off, and to induce transcription, Tc is removed.

SUMMARY OF THE INVENTION

This invention pertains to a regulatory system which utilizes componentsof the Tet repressor/operator/inducer system of prokaryotes to regulategene expression in eukaryotic cells. In particular, this inventionprovides tetracycline-regulated transcriptional inhibitors which areuseful for inhibiting expression, in a highly controlled manner, of agene linked to one or more tet operator sequences. The transcriptionalinhibitors of the invention comprise a fusion protein composed of atleast two polypeptides, a first polypeptide that binds to tet operatorsequences and a heterologous second polypeptide that directly orindirectly inhibits transcription in eukaryotic cells. The heterologousthe second polypeptide is derived from a different protein than thefirst polypeptide. Because the fusion proteins of the invention includea eukaryotic transcriptional silencer domain, they are anticipated to bemore efficient at repressing transcription in eukaryotic cells than is aTet Repressor alone.

In one embodiment of the invention, the first polypeptide of theinhibitor fusion protein binds to tet operator sequences in the absencebut not the presence of tetracycline (Tc) or an analogue thereof (e.g.,the first polypeptide is preferably a Tet repressor, such as aTn10-derived Tet repressor having an amino acid sequence shown in SEQ IDNO: 17). In the absence of tetracycline (or Tc analogue), this fusionprotein binds to tet operator sequences operatively linked to a gene ofinterest, thereby inhibiting transcription of the gene of interest. Inanother embodiment, the first polypeptide binds to tet operatorsequences in the presence but not the absence of tetracycline (e.g., thefirst polypeptide is preferably a mutated Tet repressor, such as aTn10-derived Tet repressor having an amino acid substitution at position71, 95, 101 and/or 102). Preferably, the first polypeptide has an aminoacid sequence shown in SEQ ID NO: 19. In the presence of tetracycline(or Tc analogue), this fusion protein binds to tet operator sequencesoperatively linked to a gene of interest, thereby inhibitingtranscription of the gene of interest.

The second polypeptide can be a transcriptional "silencer" domain from aprotein such as the v-erbA oncogene product, the Drosophila Krueppelprotein, the retinoic acid receptor alpha, the thyroid hormone receptoralpha, the yeast Ssn6/Tup1 protein complex, the Drosophila proteineven-skipped, SIR1, NeP1, the Drosophila dorsal protein, TSF3, SFI, theDrosophila hunchback protein, the Drosophila knirps protein, WT1,Oct-2.1, the Drosophila engrailed protein, E4BP4 or ZF5. Preferredsilencer domains include amino acid residues 403-466 of Krueppel (shownin SEQ ID NO: 21) and amino acid residues 364-635 of v-erbA (shown inSEQ ID NO: 23).

The fusion proteins of the invention may further comprise additionalpolypeptides, such as a third polypeptide which promotes transport ofthe fusion protein into a cell nucleus (i.e., a nuclear transport aminoacid sequence).

This invention further provides isolated nucleic acid molecules encodingthe transcriptional inhibitor fusion proteins of the invention andrecombinant expression vectors containing these nucleic acid moleculesin a form suitable for expression of the encoded transcriptionalinhibitor fusion protein in a host cell. The invention still furtherprovides host cells into which a recombinant expression vectors of theinvention has been introduced. Thus, a transcriptional inhibitor fusionprotein is expressed in these host cells. The host cell can be, forexample, a mammalian cell (e.g., a human cell), a yeast cell, a fungalcell or an insect cell. Moreover, the host cell can be a fertilizednon-human oocyte, in which case the host cell can be used to create atransgenic organism having cells that express the transcriptionalinhibitor fusion protein. Still further, the recombinant expressionvector can be designed to allow homologous recombination between thenucleic acid encoding the fusion protein and a target gene in a hostcell. Such homologous recombination vectors can be used to createhomologous recombinant animals that express a fusion protein of theinvention.

In a preferred embodiment, the host cells (or cells of a host organism)also contain a nucleotide sequence to be transcribed operatively linkedto at least one tet operator sequence (e.g., a gene of interest whoseexpression can be regulated by Tc or an analogue thereof). To regulatetranscription of the tet operator-linked gene of interest in these hostcells, the concentration of Tc (or analogue thereof) in contact with thehost cell is altered. For example, when the transcriptional inhibitorfusion protein binds to tet operator sequences in the absence of Tc, theconcentration of Tc in contact with the cells is decreased to therebyinhibit transcription of the tet operator-linked gene of interest (e.g.,if cells are first cultured in the presence of Tc, then Tc can beremoved from the culture medium to inhibit transcription of the gene ofinterest). Alternatively, when the transcriptional inhibitor fusionprotein binds to tet operator sequences in the presence of Tc, theconcentration of Tc in contact with the cells is increased to therebyinhibit transcription of the tet operator-linked gene of interest (e.g.,if cells are first cultured in the absence of Tc, then Tc can be addedto the culture medium to inhibit transcription of the gene of interest).

The transcriptional inhibitor fusion proteins of the invention areuseful for inhibiting gene expression in a variety of situations, asdescribed further herein. In a particularly preferred embodiment,transcription of a tet operator(tetO)-linked gene of interest isregulated by a combination of tetracycline-regulated transcriptionalinhibitor and activator fusion proteins in the same host cell to allowfor precise control of the expression level of the gene of interest. Forexample, an activator fusion protein that binds to tetO only in thepresence of Tc and an inhibitor fusion protein that binds to tetO onlyin the absence of Tc are expressed in a host cell that contains atetO-linked gene of interest. In the absence of Tc, basal levels oftranscription of the gene of interest are inhibited by the inhibitorfusion protein. Upon contact of the cell with Tc (or analogue),transcription of the gene of interest is stimulated by the activatorfusion protein. The activator and inhibitor fusion proteins of theinvention can also be used in combination to regulate the expression ofmultiple tetO-linked genes of interest.

Novel kits for regulating the expression of a gene of interest are alsowithin the scope of the invention. The kits of the invention can includea first nucleic acid molecule encoding a transcriptional inhibitorfusion protein of the invention and second nucleic acid molecule intowhich a gene of interest can be cloned such that the gene is operativelylinked to a tet operator sequence(s). The kits may further contain athird nucleic acid molecule encoding a transcriptional activator fusionprotein. Alternatively, the first and third nucleic acids (encoding thefusion proteins) may be stably incorporated into a eukaryotic host cellthat is included in the kit. Moreover, at least one tetracycline ortetracycline analogue may be included in the kits.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph depicting the stimulation of luciferase activityin HR5-C11 cells by tetracycline and different tetracycline analogues (1μg/ml f.c.). Cells were grown in the absence (-) or presence of theindicated tetracyclines for 3 days before luciferase activity wasdetermined. Each solid and hatched bar represents the luciferaseactivity of a single culture dish.

FIG. 2 is a graph depicting the relative luciferase activity in HR5-C11cells when incubated with different concentrations of doxycycline. Theresults of three independent experiments are shown.

FIG. 3 is a graph depicting the kinetics of induction of luciferaseactivity in HR5-C11 cells by doxycycline. HR5-C11 cultures were exposedto 1 μg/ml of doxycycline and luciferase activity was measured afterdifferent time intervals; () cultures containing doxycycline, (o)cultures grown in the absence of antibiotic.

FIG. 4 shows the amino acid sequences of various classes of Tetrepressors, illustrating the homology between the amino acid sequencesof different classes of Tet repressors, as compared to class B Tetrepressors (e.g., Tn10-derived). Amino acid positions in other classesof Tet repressors that are identical to class B are indicated by a dash.

FIG. 5 shows the nucleotide sequences of tet operators of differentclasses: class A (SEQ ID NO: 11), class B (SEQ ID NO: 12), class C (SEQID NO: 13), class D (SEQ ID NO: 14) and class E (SEQ ID NO: 15).

FIG. 6 is a schematic diagram of a bidirectional promoter construct forcoordinate regulation of two genes of interest operatively linked to thesame tet operators for regulation by a tetracycline-regulatedtranscriptional activator.

FIG. 7A (SEQ ID NO: 6) shows the nucleotide sequence of a bidirectionalpromoter region for coordinate regulation of two genes of interest by atetracycline-regulated transcriptional activator.

FIG. 7B (SEQ ID NO: 7) shows the nucleotide sequence of a bidirectionalpromoter region for coordinate regulation of two genes of interest by atetracycline-regulated transcriptional activator.

FIG. 8 is two graphs depicting coordinate expression of luciferase andβ-galactosidase activity by a tetracycline-regulated transcriptionalactivator.

FIG. 9A-9B are schematic diagrams of self-regulating promoters forexpression of tetracycline-regulated transcriptional activators (tTA).FIG. 9A illustrates self-regulation of expression of a wild-type Tetrepressor-containing transactivator fusion protein that binds to tetoperators in the absence of Tc. FIG. 9B illustrates self-regulation ofexpression of a mutated Tet repressor-containing transactivator fusionprotein that binds to tet operators in the presence of Tc.

FIG. 10 is a schematic diagram of the negative and positive regulationof a tet operator (tetOwt)-linked gene of interest by atetracycline-regulated transcriptional inhibitor protein (tSD) and atetracycline-inducible transcriptional activator fusion protein (rtTA),respectively, in the presence of increasing concentrations of thetetracycline analogue doxycycline.

FIG. 11 is a schematic diagram of the construction of TetR-silencerdomain fusion contructs by in-frame fusion of nucleic acid encodingeither a Krueppel or v-erbA silencer domain to the 3' end of nucleicacid encoding a Tet repressor (tetR gene).

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to nucleic acid molecules and proteins which canbe used to regulate the expression of genes in eukaryotic cells oranimals in a highly controlled manner. Regulation of gene expression bythe system of the invention involves at least two components: A genewhich is operatively linked to a regulatory sequence and a proteinwhich, in either the presence or absence of an inducible agent, binds tothe regulatory sequence and either activates or inhibits transcriptionof the gene. The system of the invention utilizes components of the Tetrepressor/operator/inducer system of prokaryotes to stimulate geneexpression in eukaryotic cells.

Various aspects of the invention pertain to fusion proteins which arecapable of either activating or inhibiting gene transcription when boundto tet operator (tetO) sequences, but which bind to tet operatorsequences only in the presence or, alternatively, in the absence oftetracycline, or an analogue thereof. Thus, in a host cell,transcription of a gene operatively linked to a tet operator sequence(s)is stimulated or inhibited by a fusion protein of the invention byaltering the concentration of tetracycline (or analogue) in contact withthe host cell (e.g., adding or removing tetracycline from a culturemedium, or administering or ceasing to administer tetracycline to a hostorganism, etc.).

The invention further pertains to target transcription units forregulation by the fusion protein of the invention. In addition toallowing for regulation of a single tet-operator linked gene ofinterest, the invention also provides novel transcription unitscontaining two or more genes to be transcribed that can be regulated ineither a coordinate or independent manner by a transactivator fusionprotein of the invention. Methods for stimulating or inhibitingtranscription of a gene using tetracycline (or analogues thereof), andkits which contain the components of the regulatory system describedherein, are also encompassed by the invention.

In the following subsections, the nucleic acids and proteins comprisingthe components of the inducible regulatory system of the invention, andtheir interrelationship, are discussed in greater detail. Thesubsections are as follows:

I. Tetracycline-Inducible Transcriptional Activators

A. The first polypeptide of the transactivator fusion protein

B. The second polypeptide of the transactivator fusion protein

C. A third polypeptide of the transactivator fusion protein

II. Expression of a Transactivator Fusion Protein

A. Expression vectors

B. Host cells

C. Introduction of nucleic acid into host cells

D. Transgenic Organisms

E. Homologous Recombinant Organisms

III. Target Transcription Units Regulated by a Tetracycline-InducibleTransactivator

A. Regulation of expression of tet operator-linked nucleotide sequences

B. Coordinate regulation of two nucleotide sequences

C. Independent regulation of multiple nucleotide sequences

D. Combined coordinate and independent regulation of multiple nucleotidesequences

IV. Tetracycline-Regulated Transcriptional Inhibitors

A. The first polypeptide of the transcritional inhibitor fusion protein

B. The second polypeptide of the transcritional inhibitor fusion protein

C. A third polypeptide of the transcritional inhibitor fusion protein

D. Expression of the transcriptional inhibitor fusion protein

V. Kits of the Invention

VI. Regulation of Gene Expression by Tetracycline or Analogues Thereof

A. Stimulation of gene expression by transactivator fusion proteins

B. Inhibition of gene expression by transcriptional inhibitor fusionproteins

C. Combined positive and negative regulation of gene expression

VII. Applications of the Invention

A. Gene Therapy

B. Production of Proteins in Vitro

C. Production of Proteins in Vivo

D. Animal Models of Human Disease

E. Production of Stable Cell Lines for Cloning

I. Tetracycline-Inducible Transcriptional Activators

In the inducible regulatory system of the invention, transcription of agene is activated by a transcriptional activator protein, also referredto herein simply as a transactivator. The transactivator of theinvention is a fusion protein. One aspect of the invention thus pertainsto fusion proteins and nucleic acids (e.g., DNA) encoding fusionproteins. The term "fusion protein" is intended to describe at least twopolypeptides, typically from different sources, which are operativelylinked. With regard to the polypeptides, the term "operatively linked"is intended to mean that the two polypeptides are connected in mannersuch that each polypeptide can serve its intended function. Typically,the two polypeptides are covalently attached through peptide bonds. Thefusion protein is preferably produced by standard recombinant DNAtechniques. For example, a DNA molecule encoding the first polypeptideis ligated to another DNA molecule encoding the second polypeptide, andthe resultant hybrid DNA molecule is expressed in a host cell to producethe fusion protein. The DNA molecules are ligated to each other in a 5'to 3' orientation such that, after ligation, the translational frame ofthe encoded polypeptides is not altered (i.e., the DNA molecules areligated to each other in-frame).

A. The first polypeptide of the transactivator fusion protein

The transactivator fusion protein of the invention is composed, in part,of a first polypeptide which binds to a tet operator sequence in thepresence of tetracycline (Tc), or an analogue thereof. The firstpolypeptide of the fusion protein is preferably a mutated Tet repressor.The term "mutated Tet repressor" is intended to include polypeptideshaving an amino acid sequence which is similar to a wild-type Tetrepressor but which has at least one amino acid difference from thewild-type Tet repressor. The term "wild-type Tet repressor" is intendedto describe a protein occurring in nature which represses transcriptionfrom tet operator sequences in prokaryotic cells in the absence of Tc.The amino acid difference(s) between a mutated Tet repressor and awild-type Tet repressor may be substitution of one or more amino acids,deletion of one or more amino acids or addition of one or more aminoacids. The mutated Tet repressor of the invention has the followingfunctional properties: 1) the polypeptide can bind to a tet operatorsequence, i.e., it retains the DNA binding specificity of a wild-typeTet repressor; and 2) it is regulated in a reverse manner bytetracycline than a wild-type Tet repressor, i.e., the mutated Tetrepressor binds to a tet operator sequence only the presence of Tc (orTc analogue) rather than in the absence of Tc.

In a preferred embodiment, a mutated Tet repressor having the functionalproperties described above is created by substitution of amino acidresidues in the sequence of a wild-type Tet repressor. For example, asdescribed in Example 1, a Tn10-derived Tet repressor having amino acidsubstitutions at amino acid positions 71, 95, 101 and 102 has thedesired functional properties and thus can be used as the firstpolypeptide in the transactivator fusion protein of the invention. Theamino acid sequence of this mutated Tet repressor is shown in SEQ ID NO:2 (positions 1-207). In one embodiment of the mutated Tet repressor,position 71 is mutated from glutamic acid to lysine, position 95 ismutated from aspartic acid to asparagine, position 101 is mutated fromleucine to serine and position 102 is mutated from glycine to asparticacid, although the invention is not limited to these particularmutations. Mutation of fewer than all four of these amino acid positionsmay be sufficient to achieve a Tet repressor with the desired functionalproperties. Accordingly, a Tet repressor is preferably mutated at atleast one of these positions. Other amino acid substitutions, deletionsor additions at these or other amino acid positions which retain thedesired functional properties of the mutated Tet repressor are withinthe scope of the invention. The crystal structure of a Tetrepressor-tetracycline complex, as described in Hinrichs, W. et al.(1994) Science 264:418-420, can be used for rational design of mutatedTet repressors. Based upon this structure, amino acid position 71 islocated outside the tetracycline binding pocket, suggesting mutation atthis site may not be necessary to achieve the desired functionalproperties of a mutated Tet repressor of the invention. In contrast,amino acid positions 95, 101 and 102 are located within the conservedtetracycline bidding pocket. Thus, the tetracycline binding pocket of aTet repressor may be targeted for mutation to create a mutated Tetrepressor of the invention.

Additional mutated Tet repressors for incorporation into a fusionprotein of the invention can be created according to the teachings ofthe invention. A number of different classes of Tet repressors have beendescribed, e.g., A, B, C, D and E (of which the Tn10-encoded repressoris a class B repressor). The amino acid sequences of the differentclasses of Tet repressors share a high degree of homology (i.e., 40-60%across the length of the proteins), including in the region encompassingthe above-described mutations. The amino acid sequences of variousclasses of Tet repressors are shown and compared in FIG. 4, and are alsodescribed in Tovar, K. et al. (1988) Mol. Gen. Genet. 215:76-80.Accordingly, equivalent mutations to those described above for theTn10-derived Tet repressor can be made in other classes of Tetrepressors for inclusion in a fusion protein of the invention. Forexample, amino acid position 95, which is an aspartic acid in all fiverepressor classes, can be mutated to asparagine in any class ofrepressor. Similarly, position 102, which is glycine in all fiverepressor classes, can be mutated to aspartic acid in any class ofrepressor. Additional suitable equivalent mutations will be apparent tothose skilled in the art and can be created and tested for functionalityby procedures described herein. Nucleotide and amino acid sequences ofTet repressors of the A, C, D and E classes are disclosed in Waters, S.H. et al. (1983) Nucl. Acids Res 11:6089-6105, Unger, B. et al. (1984)Gene 3: 103-108, Unger, B. et al. (1984) Nucl Acids Res. 12:7693-7703and Tovar, K. et al. (1988) Mol. Gen. Genet. 215:76-80, respectively.These wild-type sequences can be mutated according to the teachings ofthe invention for use in the inducible regulatory system describedherein.

Alternative to the above-described mutations, additional suitablemutated Tet repressors (i.e., having the desired functional propertiesdescribed above) can be created by mutagenesis of a wild type Tetrepressor and selection as described in Example 1. The nucleotide andamino acid sequences of wild-type class B Tet repressors are disclosedin Hillen, W. and Schollmeier, K. (1983) Nucl. Acids Res. 11:525-539 andPostle, K. et al. (1984) Nucl. Acids Res. 12:4849-4863. The nucleotideand amino acid sequences of wild-type class A, C, D and E typerepressors are cited above. A mutated Tet repressor can be created andselected, for example as follows: a nucleic acid (e.g., DNA) encoding awild-type Tet repressor is subjected to random mutagenesis and theresultant mutated nucleic acids are incorporated into an expressionvector and introduced into a host cell for screening. A screening assayis used which allows for selection of a Tet repressor which binds to atet operator sequence only in the presence of tetracycline. For example,a library of mutated nucleic acids in an expression vector can beintroduced into an E. coli strain in which tet operator sequencescontrol the expression of a gene encoding a Lac repressor and the Lacrepressor controls the expression of a gene encoding an selectablemarker (e.g., drug resistance). Binding of a Tet repressor to tetoperator sequences in the bacteria will inhibit expression of the Lacrepressor, thereby inducing expression of the selectable marker gene.Cells expressing the marker gene are selected based upon the selectablephenotype (e.g., drug resistance). For wild-type Tet repressors,expression of the selectable marker gene will occur in the absence ofTc. A nucleic acid encoding a mutated Tet repressor is selected usingthis system based upon the ability of the nucleic acid to induceexpression of the selectable marker gene in the bacteria only in thepresence of Tc.

A first polypeptide of the transactivator fusion protein (e.g., themutated Tet repressor) has the property of binding specifically to a tetoperator sequence. Each class of Tet repressor has a correspondingtarget tet operator sequence. Accordingly, the term "tet operatorsequence" is intended to encompass all classes of tet operatorsequences, e.g. class A, B, C, D, and E. Nucleotide sequences of thesefive classes of tet operators are shown in FIG. 5 and SEQ ID NOs: 11-15,and are described in Waters, S. H. et al. (1983) cited supra, Hillen, W.and Schollenmeier, K. (1983) cited supra, Stuber, D. and Bujard, H.(1981) Proc. Natl. Acad. Sci. USA 78:167-171, Unger, B. et al. (1984)cited supra and Tovar, K. et al. (1988) cited supra. In a preferredembodiment, the mutated Tet repressor is a Tn10-encoded repressor (i.e.,class B) and the tet operator sequence is a class B tet operatorsequence. Alternatively, a mutated class A Tet repressor can be usedwith a class A tet operator sequence, and so on for the other classes ofTet repressor/operators.

Another approach for creating a mutated Tet repressor which binds to aclass A tet operator is to further mutate the already mutatedTn10-derived Tet repressor described herein (a class B repressor) suchthat it no longer binds efficiently to a class B type operator butinstead binds efficiently to a class A type operator. It has been foundthat nucleotide position 6 of class A or B type operators is thecritical nucleotide for recognition of the operator by its complimentaryrepressor (position 6 is a G/C pair in class B operators and an A/T pairin class A operators) (see Wissman et al. (1988) J. Mol. Biol.202:397-406). It has also been found that amino acid position 40 of aclass A or class B Tet repressor is the critical amino acid residue forrecognition of position 6 of the operator (amino acid position 40 is athreonine in class B repressors but is an alanine in class Arepressors). It still further has been found that substitution of Thr40of a class B repressor with Ala alters its binding specificity such thatthe repressor can now bind a class A operator (similarly, substitutionof Ala4O of a class A repressor with Thr alters its binding specificitysuch that the repressor can now bind a class B operator) (see Altschmiedet al. (1988) EMBO J. 7:4011-4017). Accordingly, one can alter thebinding specificity of the mutated Tn10-derived Tet repressor disclosedherein by additionally changing amino acid residue 40 from Thr to Ala bystandard molecular biology techniques (e.g., site directed mutagenesis).

A mutated Tet repressor having specific mutations (e.g., at positions71, 95, 101 and/or 102, as described above) can be created byintroducing nucleotide changes into a nucleic acid encoding a wild-typerepressor by standard molecular biology techniques, e.g. site directedmutagenesis or PCR-mediated mutagenesis using oligonucleotide primersincorporating the nucleotide mutations. Alternatively, when a mutatedTet repressor is identified by selection from a library, the mutatednucleic acid can be recovered from the library vector. To create atransactivator fusion protein of the invention, a nucleic acid encodinga mutated Tet repressor is then ligated in-frame to another nucleic acidencoding a transcriptional activation domain and the fusion construct isincorporated into a recombinant expression vector. The transactivatorfusion protein can be expressed by introducing the recombinantexpression vector into a host cell or animal.

B. The second polypeptide of the transactivator fusion protein

The first polypeptide of the transactivator fusion protein isoperatively linked to a second polypeptide which directly or indirectlyactivates transcription in eukaryotic cells. To operatively link thefirst and second polypeptides, typically nucleotide sequences encodingthe first and second polypeptides are ligated to each other in-frame tocreate a chimeric gene encoding a fusion protein, although the first andsecond polypeptides can be operatively linked by other means thatpreserve the function of each polypeptide (e.g., chemicallycrosslinked). In a preferred embodiment, the second polypeptide of thetransactivator itself possesses transcriptional activation activity(i.e., the second polypeptide directly activates transcription). Inanother embodiment, the second polypeptide activates transcription by anindirect mechanism, through recruitment of a transcriptional activationprotein to interact with the fusion protein. Accordingly, the term "apolypeptide which activates transcription in eukaryotic cells" as usedherein is intended to include polypeptides which either directly orindirectly activates transcription.

Polypeptides which can function to activate transcription in eukaryoticcells are well known in the art. In particular, transcriptionalactivation domains of many DNA binding proteins have been described andhave been shown to retain their activation function when the domain istransferred to a heterologous protein. A preferred polypeptide for usein the fusion protein of the invention is the herpes simplex virusvirion protein 16 (referred to herein as VP16, the amino acid sequenceof which is disclosed in Triezenberg, S. J. et al. (1988) Genes Dev.2:718-729). In one embodiment, about 127 of the C-terminal amino acidsof VP16 are used. For example, a polypeptide having an amino acidsequence shown in SEQ ID NO: 2 (positions 208-335) can be used as thesecond polypeptide in the fusion protein. In another embodiment, atleast one copy of about 11 amino acids from the C-terminal region ofVP16 which retain transcriptional activation ability is used as thesecond polypeptide. Preferably, a dimer of this region (i.e., about 22amino acids) is used. Suitable C-terminal peptide portions of VP16 aredescribed in Seipel, K. et al. (EMBO J. (1992) 13:4961-4968). Forexample, a dimer of a peptide having an amino acid sequence shown in SEQID NO: 4 (encoded by a nucleotide sequence shown in SEQ ID NO: 3) can beused as the second polypeptide in the fusion protein.

Other polypeptides with transcriptional activation ability in eukaryoticcells can be used in the fusion protein of the invention.Transcriptional activation domains found within various proteins havebeen grouped into categories based upon similar structural features.Types of transcriptional activation domains include acidic transcriptionactivation domains, proline-rich transcription activation domains,serine/threonine-rich transcription activation domains andglutamine-rich transcription activation domains. Examples of acidictranscriptional activation domains include the VP16 regions alreadydescribed and amino acid residues 753-881 of GAL4. Examples ofproline-rich activation domains include amino acid residues 399-499 ofCTF/NF1 and amino acid residues 31-76 of AP2. Examples ofserine/threonine-rich transcription activation domains include aminoacid residues 1-427 of ITF1 and amino acid residues 2-451 of ITF2.Examples of glutamine-rich activation domains include amino acidresidues 175-269 of Oct1 and amino acid residues 132-243 of Sp1. Theamino acid sequences of each of the above described regions, and ofother useful transcriptional activation domains, are disclosed inSeipel, K. et al. (EMBO J. (1992) 12:4961-4968).

In addition to previously described transcriptional activation domains,novel transcriptional activation domains, which can be identified bystandard techniques, are within the scope of the invention. Thetranscriptional activation ability of a polypeptide can be assayed bylinking the polypeptide to another polypeptide having DNA bindingactivity and determining the amount of transcription of a targetsequence that is stimulated by the fusion protein. For example, astandard assay used in the art utilizes a fusion protein of a putativetranscriptional activation domain and a GAL4 DNA binding domain (e.g.,amino acid residues 1-93). This fusion protein is then used to stimulateexpression of a reporter gene linked to GAL4 binding sites (see e.g.,Seipel, K et al. (1992) EMBO J. 11:4961-4968 and references citedtherein).

In another embodiment, the second polypeptide of the fusion proteinindirectly activates transcription by recruiting a transcriptionalactivator to interact with the fusion protein. For example, a mutatedtetR of the invention can be fused to a polypeptide domain (e.g., adimerization domain) capable of mediating a protein-protein interactionwith a transcriptional activator protein, such as an endogenousactivator present in a host cell. It has been demonstrated thatfunctional associations between DNA binding domains and transactivationdomains need not be covalent (see e.g., Fields and Song (1989) Nature340:245-247; Chien et al. (1991) Proc. Natl. Acad. Sci. USA88:9578-9582; Gyuris et al. (1993) Cell 75:791-803; and Zervos, A. S.(1993) Cell 72:223-232). Accordingly, the second polypeptide of thefusion protein may not directly activate transcription but rather mayform a stable interaction with an endogenous polypeptide bearing acompatible protein-protein interaction domain and transactivationdomain. Examples of suitable interaction (or dimerization) domainsinclude leucine zippers (Landschulz et al. (1989) Science243:1681-1688), helix-loop-helix domains (Murre, C. et al. (1989) Cell58:537-544) and zinc finger domains (Frankel, A. D. et al. (1988)Science 240:70-73). Interaction of a dimerization domain present in thefusion protein with an endogeneous nuclear factor results in recruitmentof the transactivation domain of the nuclear factor to the fusionprotein, and thereby to a tet operator sequence to which the fusionprotein is bound.

C. A third polypeptide of the transactivator fusion protein

In addition to a mutated Tet repressor and a transcriptional activationdomain, a fusion protein of the invention can contain an operativelylinked third polypeptide which promotes transport of the fusion proteinto a cell nucleus. Amino acid sequences which, when included in aprotein, function to promote transport of the protein to the nucleus areknown in the art and are termed nuclear localization signals (NLS).Nuclear localization signals typically are composed of a stretch ofbasic amino acids. When attached to a heterologous protein (e.g., afusion protein of the invention), the nuclear localization signalpromotes transport of the protein to a cell nucleus. The nuclearlocalization signal is attached to a heterologous protein such that itis exposed on the protein surface and does not interfere with thefunction of the protein. Preferably, the NLS is attached to one end ofthe protein, e.g. the N-terminus. The amino acid sequence of anon-limiting example of an NLS that can be included in a fusion proteinof the invention is shown in SEQ ID NO: 5. Preferably, a nucleic acidencoding the nuclear localization signal is spliced by standardrecombinant DNA techniques in-frame to the nucleic acid encoding thefusion protein (e.g., at the 5' end).

The plasmid pUHD17-1 (described in further detail in Example 1), whichcomprises a transactivator of the invention having the nucleotidesequence shown in SEQ ID NO: 1, has been deposited on July 8, 1994 underthe provisions of the Budapest Treaty at the Deutsche Sammlung VonMikroorganismen und ZellKulturen GmbH (DSM) in Braunschweig, Germany andassigned deposit number DSM 9279.

II. Expression of a Transactivator Fusion Protein

A. Expression Vectors

A nucleic acid of the invention encoding a transactivator fusionprotein, as described above, can be incorporated into a recombinantexpression vector in a form suitable for expression of the fusionprotein in a host cell. The term "in a form suitable for expression ofthe fusion protein in a host cell" is intended to mean that therecombinant expression vector includes one or more regulatory sequencesoperatively linked to the nucleic acid encoding the fusion protein in amanner which allows for transcription of the nucleic acid into mRNA andtranslation of the mRNA into the fusion protein. The term "regulatorysequence" is art-recognized and intended to include promoters, enhancersand other expression control elements (e.g., polyadenylation signals).Such regulatory sequences are known to those skilled in the art and aredescribed in Goeddel, Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990). It should be understoodthat the design of the expression vector may depend on such factors asthe choice of the host cell to be transfected and/or the amount offusion protein to be expressed.

When used in mammalian cells, a recombinant expression vector's controlfunctions are often provided by viral genetic material. For example,commonly used promoters are derived from polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40. Use of viral regulatory elements todirect expression of the fusion protein can allow for high levelconstitutive expression of the fusion protein in a variety of hostcells. In a preferred recombinant expression vector, the sequencesencoding the fusion protein are flanked upstream (i.e., 5') by the humancytomegalovirus IE promoter and downstream (i.e., 3') by an SV40 poly(A)signal. For example, an expression vector similar to that described inExample 1 can be used. The human cytomegalovirus IE promoter isdescribed in Boshart et al. (1985) Cell 41:521-530. Other ubiquitouslyexpressing promoters which can be used include the HSV-Tk promoter(disclosed in McKnight et al. (1984) Cell 37:253-262) and β-actinpromoters (e.g., the human β-actin promoter as described by Ng et al.(1985) Mol. Cell. Biol 5:2720-2732).

Alternatively, the regulatory sequences of the recombinant expressionvector can direct expression of the fusion protein preferentially in aparticular cell type, i.e., tissue-specific regulatory elements can beused. Non-limiting examples of tissue-specific promoters which can beused include the albumin promoter (liver-specific; Pinkert et al. (1987)Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton(1988) Adv. Immunol. 43:235-275), in particular promoters of T cellreceptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

Alternatively, a self-regulating construct encoding a transactivatorfusion protein can be created. To accomplish this, nucleic acid encodingthe fusion protein is operatively linked to a minimal promoter sequenceand at least one tet operator sequence. For example, the nucleic acid ofSEQ ID NO: 1 can be linked to a promoter having a nucleotide sequenceshown in SEQ ID NO: 8, 9 or 10 (the nucleic acids of SEQ ID NOs: 8 and 9comprise a minimal CMV promoter and ten tet operators; the nucleic acidsof SEQ ID NO: 10 comprises a TK promoter and ten tet operators). Aschematic diagram of such a self-regulating construct is shown in FIG.9B. When this nucleic acid is introduced into a cell (e.g., in arecombinant expression vector), a small amount of basal transcription ofthe transactivator gene is likely to occur due to "leakiness". In thepresence of Tc (or analogue thereof) this small amount of thetransactivator fusion protein will bind to the tet operator sequence(s)upstream of the nucleotide sequence encoding the transactivator andstimulate additional transcription of the nucleotide sequence encodingthe transactivator, thereby leading to further production of thetransactivator fusion protein in the cell. It will be appreciated bythose skilled in the art that such a self-regulating promoter can alsobe used in conjunction with other tetracycline-regulatedtransactivators, such as the wild-type Tet repressor fusion protein(tTA) described in Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad.Sci. USA 89:5547-5551, which binds to tet operators in the absence of Tc(as illustrated in FIG. 9A). When used in conjunction with thistransactivator, self-regulated transcription of the nucleotide sequenceencoding this transactivator is stimulated in the absence of Tc. Theplasmid pUHD15-3, which comprises nucleotide sequences encoding the tTAdescribed in Gossen and Bujard (1992), cited supra, operatively linkedto a self-regulating promoter, has been deposited on July 8, 1994 underthe provisions of the Budapest Treaty at the Deutsche Sammlung VonMikroorganismen und ZellKulturen GmbH (DSM) in Braunschweig, Germany andassigned deposit number DSM 9280.

In one embodiment, the recombinant expression vector of the invention isa plasmid, such as that described in Example 1. Alternatively, arecombinant expression vector of the invention can be a virus, orportion thereof, which allows for expression of a nucleic acidintroduced into the viral nucleic acid. For example, replicationdefective retroviruses, adenoviruses and adeno-associated viruses can beused. Protocols for producing recombinant retroviruses and for infectingcells in vitro or in vivo with such viruses can be found in CurrentProtocols in Molecular Biology, Ausubel, F. M. et al. (eds.) GreenePublishing Associates, (1989), Sections 9.10-9.14 and other standardlaboratory manuals. Examples of suitable retroviruses include pLJ, pZIP,pWE and pEM which are well known to those skilled in the art. Examplesof suitable packaging virus lines include ψCrip, ψCre, ψ2 and ψAm. Thegenome of adenovirus can be manipulated such that it encodes andexpresses a transactivator fusion protein but is inactivated in terms ofits ability to replicate in a normal lytic viral life cycle. See forexample Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al.(1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155.Suitable adenoviral vectors derived from the adenovirus strain Ad type 5d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are wellknown to those skilled in the art. Alternatively, an adeno-associatedvirus vector such as that described in Tratschin et al. (1985) Mol.Cell. Biol. 5:3251-3260 can be used to express a transactivator fusionprotein.

B. Host Cells

A fusion protein of the invention is expressed in a eukaryotic cell byintroducing nucleic acid encoding the fusion protein into a host cell,wherein the nucleic acid is in a form suitable for expression of thefusion protein in the host cell. For example, a recombinant expressionvector of the invention, encoding the fusion protein, is introduced intoa host cell. Alternatively, nucleic acid encoding the fusion proteinwhich is operatively linked to regulatory sequences (e.g., promotersequences) but without additional vector sequences can be introducedinto a host cell. As used herein, the term "host cell" is intended toinclude any eukaryotic cell or cell line so long as the cell or cellline is not incompatible with the protein to be expressed, the selectionsystem chosen or the fermentation system employed. Non-limiting examplesof mammalian cell lines which can be used include CHO dhfr⁻ cells(Urlaub and Chasin (1980) Proc. Natl. Acad Sci. USA 77:4216-4220), 293cells (Graham et al. (1977) J. Gen. Virol. 36: pp 59) or myeloma cellslike SP2 or NS0 (Galfre and Milstein (1981) Meth. Enzymol. 73(B):3-46).

In addition to cell lines, the invention is applicable to normal cells,such as cells to be modified for gene therapy purposes or embryoniccells modified to create a transgenic or homologous recombinant animal.Examples of cell types of particular interest for gene therapy purposesinclude hematopoietic stem cells, myoblasts, hepatocytes, lymphocytes,neuronal cells and skin epithelium and airway epithelium. Additionally,for transgenic or homologous recombinant animals, embryonic stem cellsand fertilized oocytes can be modified to contain nucleic acid encodinga transactivator fusion protein. Moreover, plant cells can be modifiedto create transgenic plants.

The invention is broadly applicable and encompasses non-mammalianeukaryotic cells as well, including insect (e.g,. Sp. frugiperda), yeast(e.g., S. cerevisiae, S. pombe, P. pastoris, K. lactis, H. polymorpha;as generally reviewed by Fleer, R. (1992) Current Opinion inBiotechnology 3(5):486-496)), fungal and plant cells. Examples ofvectors for expression in yeast S. cerivisae include pYepSec1 (Baldari,et al., (1987) Embo J. 6:229-234), pMFa (Kujan and Herskowitz, (1982)Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), andpYES2 (Invitrogen Corporation, San Diego, Calif.). The fusion proteincan be expressed in insect cells using baculovirus expression vectors(e.g., as described in O'Reilly et al. (1992) Baculovirus ExpressionVectors: A Laboratory Manual, Stockton Press). Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith et al., (1983) Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow, V. A., and Summers, M. D.,(1989) Virology 170:31-39).

C. Introduction of Nucleic Acid into a Host Cell

Nucleic acid encoding the fusion protein can be introduced into a hostcell by standard techniques for transfecting eukaryotic cells. The term"transfecting" or "transfection" is intended to encompass allconventional techniques for introducing nucleic acid into host cells,including calcium phosphate co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, electroporation and microinjection. Suitablemethods for transfecting host cells can be found in Sambrook et al.(Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring HarborLaboratory press (1989)), and other laboratory textbooks.

The number of host cells transformed with a nucleic acid of theinvention will depend, at least in part, upon the type of recombinantexpression vector used and the type of transfection technique used.Nucleic acid can be introduced into a host cell transiently, or moretypically, for long term regulation of gene expression, the nucleic acidis stably integrated into the genome of the host cell or remains as astable episome in the host cell. Plasmid vectors introduced intomammalian cells are typically integrated into host cell DNA at only alow frequency. In order to identify these integrants, a gene thatcontains a selectable marker (e.g., drug resistance) is generallyintroduced into the host cells along with the nucleic acid of interest.Preferred selectable markers include those which confer resistance tocertain drugs, such as G418 and hygromycin. Selectable markers can beintroduced on a separate plasmid from the nucleic acid of interest or,are introduced on the same plasmid. Host cells transfected with anucleic acid of the invention (e.g., a recombinant expression vector)and a gene for a selectable marker can be identified by selecting forcells using the selectable marker. For example, if the selectable markerencodes a gene conferring neomycin resistance, host cells which havetaken up nucleic acid can be selected with G418. Cells that haveincorporated the selectable marker gene will survive, while the othercells die.

A host cell transfected with a nucleic acid encoding a fusion protein ofthe invention can be further transfected with one or more nucleic acidswhich serve as the target for the fusion protein. The target nucleicacid comprises a nucleotide sequence to be transcribed operativelylinked to at least one tet operator sequence (described in more detailin Section III below).

Nucleic acid encoding the fusion protein of the invention can beintroduced into eukaryotic cells growing in culture in vitro byconventional transfection techniques (e.g., calcium phosphateprecipitation, DEAE-dextran transfection, electroporation etc.). Nucleicacid can also be transferred into cells in vivo, for example byapplication of a delivery mechanism suitable for introduction of nucleicacid into cells in vivo, such as retroviral vectors (see e.g., Ferry, Net al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; and Kay, M. A. etal. (1992) Human Gene Therapy 3:641-647), adenoviral vectors (see e.g.,Rosenfeld, M. A. (1992) Cell 68:143-155; and Herz, J. and Gerard, R. D.(1993) Proc. Natl. Acad. Sci. USA 90:2812-2816), receptor-mediated DNAuptake (see e.g., Wu, G. and Wu, C. H. (1988) J. Biol. Chem. 263:14621;Wilson et al. (1992) J. Biol. Chem. 267:963-967; and U.S. Pat. No.5,166,320), direct injection of DNA (see e.g., Acsadi et al. (1991)Nature 332: 815-818; and Wolff et al. (1990) Science 247:1465-1468) orparticle bombardment (see e.g., Cheng, L. et al. (1993) Proc. Natl.Acad. Sci. USA 90:4455-4459; and Zelenin, A. V. et al. (1993) FEBSLetters 315:29-32). Thus, for gene therapy purposes, cells can bemodified in vitro and administered to a subject or, alternatively, cellscan be directly modified in vivo.

D. Transgenic Organisms

Nucleic acid a transactivator fusion protein can transferred into afertilized oocyte of a non-human animal to create a transgenic animalwhich expresses the fusion protein of the invention in one or more celltypes. A transgenic animal is an animal having cells that contain atransgene, wherein the transgene was introduced into the animal or anancestor of the animal at a prenatal, e.g., an embryonic, stage. Atransgene is a DNA which is integrated into the genome of a cell fromwhich a transgenic animal develops and which remains in the genome ofthe mature animal, thereby directing the expression of an encoded geneproduct in one or more cell types or tissues of the transgenic animal.In one embodiment, the non-human animal is a mouse, although theinvention is not limited thereto. In other embodiments, the transgenicanimal is a goat, sheep, pig, cow or other domestic farm animal. Suchtransgenic animals are useful for large scale production of proteins (socalled "gene pharming").

A transgenic animal can be created, for example, by introducing anucleic acid encoding the fusion protein (typically linked toappropriate regulatory elements, such as a constitutive ortissue-specific enhancer) into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, and allowing the oocyte to develop in apseudopregnant female foster animal. Intronic sequences andpolyadenylation signals can also be included in the transgene toincrease the efficiency of expression of the transgene. Methods forgenerating transgenic animals, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866 and 4,870,009 and Hogan, B. et al., (1986) ALaboratory Manual, Cold Spring Harbor, New York, Cold Spring HarborLaboratory. A transgenic founder animal can be used to breed additionalanimals carrying the transgene. Transgenic animals carrying a transgeneencoding the fusion protein of the invention can further be bred toother transgenic animals carrying other transgenes, e.g., to atransgenic animal which contains a gene operatively linked to a tetoperator sequence (discussed in more detail in Section III below).

It will be appreciated that, in addition to transgenic animals, theregulatory system described herein can be applied to other transgenicorganisms, such as transgenic plants. Transgenic plants can be made byconventional techniques known in the art. Accordingly, the inventionencompasses non-human transgenic organisms, including animals andplants, that contains cells which express the transactivator fusionprotein of the invention (i.e., a nucleic acid encoding thetransactivator is incorporated into one or more chromosomes in cells ofthe transgenic organism).

E. Homologous Recombinant Organisms

The invention also provides a homologous recombinant non-human organismexpressing the fusion protein of the invention. The term "homologousrecombinant organism" as used herein is intended to describe anorganism, e.g. animal or plant, containing a gene which has beenmodified by homologous recombination between the gene and a DNA moleculeintroduced into a cell of the animal, e.g., an embryonic cell of theanimal. In one embodiment, the non-human animal is a mouse, although theinvention is not limited thereto. An animal can be created in whichnucleic acid encoding the fusion protein has been introduced into aspecific site of the genome, i.e., the nucleic acid has homologouslyrecombined with an endogenous gene.

To create such a homologous recombinant animal, a vector is preparedwhich contains DNA encoding the fusion protein flanked at its 5' and 3'ends by additional nucleic acid of a eukaryotic gene at which homologousrecombination is to occur. The additional nucleic acid flanking thatencoding the fusion protein is of sufficient length for successfulhomologous recombination with the eukaryotic gene. Typically, severalkilobases of flanking DNA (both at the 5' and 3' ends) are included inthe vector (see e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell51:503 for a description of homologous recombination vectors). Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced DNA has homologouslyrecombined with the endogenous DNA are selected (see e.g., Li, E. et al.(1992) Cell 69:915). The selected cells are then injected into ablastocyst of an animal (e.g., a mouse) to form aggregation chimeras(see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: APractical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987) pp.113-152). A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term.Progeny harbouring the homologously recombined DNA in their germ cellscan be used to breed animals in which all cells of the animal containthe homologously recombined DNA. These "germline transmission" animalscan further be mated to animals carrying a gene operatively linked to atleast one tet operator sequence (discussed in more detail in Section IIIbelow).

In addition to the homologous recombination approaches described above,enzyme-assisted site-specific integration systems are known in the artand can be applied to the components of the regulatory system of theinvention to integrate a DNA molecule at a predetermined location in asecond target DNA molecule. Examples of such enzyme-assisted integrationsystems include the Cre recombinase-lox target system (e.g., asdescribed in Baubonis, W. and Sauer, B. (1993) Nucl. Acids Res.21:2025-2029; and Fukushige, S. and Sauer, B. (1992) Proc. Natl. Acad.Sci. USA 89:7905-7909) and the FLP recombinase-FRT target system (e.g.,as described in Dang, D. T. and Perrimon, N. (1992) Dev. Genet.13:367-375; and Fiering, S. et al. (1993) Proc. Natl. Acad. Sci. USA90:8469-8473).

III. Target Transcription Units Regulated by a Tetracycline-InducibleTransactivator

A fusion protein of the invention is used to regulate the transcriptionof a target nucleotide sequence. This target nucleotide sequence isoperatively linked to a regulatory sequence to which the fusion proteinbinds. More specifically, the fusion protein regulates expression of anucleotide sequence operatively linked to at least one tet operatorsequence. Accordingly, another aspect of the invention relates to targetnucleic acids (e.g., DNA molecules) comprising a nucleotide sequence tobe transcribed operatively linked to at least one tet operator sequence.Such nucleic acids are also referred to herein as tet-regulatedtranscription units (or simply transcription units).

Within a transcription unit, the "nucleotide sequence to be transcribed"typically includes a minimal promoter sequence which is not itselftranscribed but which serves (at least in part) to position thetranscriptional machinery for transcription. The minimal promotersequence is linked to the transcribed sequence in a 5' to 3' directionby phosphodiester bonds (i.e., the promoter is located upstream of thetranscribed sequence) to form a contiguous nucleotide sequence.Accordingly, as used herein, the terms "nucleotide sequence to betranscribed" or "target nucleotide sequence" are intended to includeboth the nucleotide sequence which is transcribed into mRNA and anoperatively linked upstream minimal promoter sequence. The term "minimalpromoter" is intended to describe a partial promoter sequence whichdefines the start site of transcription for the linked sequence to betranscribed but which by itself is not capable of initiatingtranscription efficiently, if at all. Thus, the activity of such aminimal promoter is dependent upon the binding of a transcriptionalactivator (such as the tetracycline-inducible fusion protein of theinvention) to an operatively linked regulatory sequence (such as one ormore tet operator sequences). In one embodiment, the minimal promoter isfrom the human cytomegalovirus (as described in Boshart et al. (1985)Cell 41:521-530). Preferably, nucleotide positions between about +75 to-53 and +75 to -31 are used. Other suitable minimal promoters are knownin the art or can be identified by standard techniques. For example, afunctional promoter which activates transcription of a contiguouslylinked reporter gene (e.g., chloramphenicol acetyl transferase,β-galactosidase or luciferase) can be progressively deleted until it nolonger activates expression of the reporter gene alone but ratherrequires the presence of an additional regulatory sequence(s).

Within a transcription unit, the target nucleotide sequence (includingthe transcribed nucleotide sequence and its upstream minimal promotersequence) is operatively linked to at least one tet operator sequence.In a typical configuration, the tet operator sequence(s) is operativelylinked upstream (i.e., 5') of the minimal promoter sequence through aphosphodiester bond at a suitable distance to allow for transcription ofthe target nucleotide sequence upon binding of a regulatory protein(e.g., the transactivator fusion protein) to the tet operator sequence.That is, the transcription unit is comprised of, in a 5' to 3'direction: tet operator sequence(s)--a minimal promoter--a transcribednucleotide sequence. It will be appreciated by those skilled in the artthat there is some flexibility in the permissible distance between thetet operator sequence(s) and the minimal promoter, although typicallythe tet operator sequences will be located within about 200-400 basepairs upstream of the minimal promoter.

The nucleotide sequences of examples of tet-regulated promoters,containing tet operator sequences linked to a minimal promoter, that canbe used in the invention are shown in SEQ ID NO: 8-10. The nucleotidesequences of SEQ ID NOs: 8 and 9 comprise a cytomegalovirus minimalpromoter linked to ten tet operator sequences; the two nucleotidesequences differ in the distance between the operators and the firsttranscribed nucleotide. The nucleotide sequence of SEQ ID NO: 10comprises a herpes simplex virus minimal tk promoter linked to ten tetoperator sequences. The promoter of SEQ ID NO: 8 corresponds to P_(hCMV)*-1, described in Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad.Sci. USA 89:5547-5551. The promoter of SEQ ID NO: 9 corresponds toP_(hCMV) *-2, also described in Gossen, M. and Bujard, H, cited supra.

Alternatively, since regulatory elements have been observed in the artto function downstream of sequences to be transcribed, it is likely thatthe tet operator sequence(s) can be operatively linked downstream (i.e.,3') of the transcribed nucleotide sequence. Thus, in this configuration,the transcription unit is comprised of, in a 5' to 3' direction: aminimal promoter--a transcribed nucleotide sequence--tet operatorsequence(s). Again, it will be appreciated that there is likely to besome flexibility in the permissible distance downstream at which the tetoperator sequence(s) can be linked.

The term "tet operator sequence" is intended to encompass all classes oftet operators (e.g., A, B, C, D and E). A nucleotide sequence to betranscribed can be operatively linked to a single tet operator sequence,or for an enhanced range of regulation, it can be operatively linked tomultiple tet operator sequences (e.g., two, three, four, five, six,seven, eight, nine, ten or more operator sequences). In a preferredembodiment, the sequence to be transcribed is operatively linked toseven tet operator sequences.

A tet-regulated transcription unit can further be incorporated into arecombinant vector (e.g., a plasmid or viral vector) by standardrecombinant DNA techniques. The transcription unit, or recombinantvector in which it is contained, can be introduced into a host cell bystandard transfection techniques, such as those described above. Itshould be appreciated that, after introduction of the transcription unitinto a population of host cells, it may be necessary to select a hostcell clone which exhibit low basal expression of the tet operator-linkednucleotide sequence (i.e., selection for a host cell in which thetranscription unit has integrated at a site that results in low basalexpression of the tet operator-linked nucleotide sequence). Furthermore,a tet-regulated transcription unit can be introduced, by proceduresdescribed above, into the genome of a non-human animal at an embryonicstage or into plant cells to create a transgenic or homologousrecombinant organism carrying the transcription unit in some or all ofits cells. Again, it should be appreciated that it may be necessary toselect a transgenic or homologous organism in which there is low basalexpression of the tet operator-linked nucleotide sequence in cells ofinterest.

In one embodiment, the target nucleotide sequence of the tet-regulatedtranscription unit encodes a protein of interest. Thus, upon inductionof transcription of the nucleotide sequence by the transactivator of theinvention and translation of the resultant mRNA, the protein of interestis produced in a host cell or animal. Alternatively, the nucleotidesequence to be transcribed can encode for an active RNA molecule, e.g.,an antisense RNA molecule or ribozyme. Expression of active RNAmolecules in a host cell or animal can be used to regulate functionswithin the host (e.g., prevent the production of a protein of interestby inhibiting translation of the mRNA encoding the protein).

A transactivator of the invention can be used to regulate transcriptionof an exogenous nucleotide sequence introduced into the host cell oranimal. An "exogenous" nucleotide sequence is a nucleotide sequencewhich is introduced into the host cell and typically is inserted intothe genome of the host. The exogenous nucleotide sequence may not bepresent elsewhere in the genome of the host (e.g., a foreign nucleotidesequence) or may be an additional copy of a sequence which is presentwithin the genome of the host but which is integrated at a differentsite in the genome. An exogenous nucleotide sequence to be transcribedand an operatively linked tet operator sequence(s) can be containedwithin a single nucleic acid molecule which is introduced into the hostcell or animal.

Alternatively, a transactivator of the invention can be used to regulatetranscription of an endogenous nucleotide sequence to which a tetoperator sequence(s) has been linked. An "endogenous" nucleotidesequence is a nucleotide sequence which is present within the genome ofthe host. An endogenous gene can be operatively linked to a tet operatorsequence(s) by homologous recombination between a tetO-containingrecombination vector and sequences of the endogeneous gene. For example,a homologous recombination vector can be prepared which includes atleast one tet operator sequence and a miminal promoter sequence flankedat its 3' end by sequences representing the coding region of theendogenous gene and flanked at its 5' end by sequences from the upstreamregion of the endogenous gene by excluding the actual promoter region ofthe endogenous gene. The flanking sequences are of sufficient length forsuccessful homologous recombination of the vector DNA with theendogenous gene. Preferably, several kilobases of flanking DNA areincluded in the homologous recombination vector. Upon homologousrecombination between the vector DNA and the endogenous gene in a hostcell, a region of the endogenous promoter is replaced by the vector DNAcontaining one or more tet operator sequences operably linked to aminimal promoter. Thus, expression of the endogenous gene is no longerunder the control of its endogenous promoter but rather is placed underthe control of the tet operator sequence(s) and the minimal promoter.

In another embodiment, tet operator sequences can be inserted elsewherewithin an endogenous gene, preferably within a 5' or 3' regulatoryregion, via homologous recombination to create an endogenous gene whoseexpression can be regulated by a tetracycline-regulated fusion proteindescribed herein. For example, one or more tetO sequences can beinserted into a promoter or enhancer region of an endogenous gene suchthat promoter or enhancer function is maintained (i.e., the tetOsequences are introduced into a site of the promoter/enhancer regionthat is not critical for promoter/enhancer function). Regions withinpromoters or enhancers which can be altered without loss ofpromoter/enhancer function are known in the art for many genes or can bedetermined by standard techniques for analyzing critical regulatoryregions. An endogenous gene having tetO sequences inserted into anon-critical regulatory region will retain the ability to be expressedin its normal constitutive and/or tissue-specific manner but,additionally, can be downregulated by a tetracycline-controlledtranscriptional inhibitor protein in a controlled manner. For example,constitutive expression of such a modified endogenous gene can beinhibited by in the presence of tetracycline (or analogue) using aninhibitor fusion protein that binds to tetO sequences in the presence oftetracycline (or analogue) (as described in further detail in Section IVand Section VI, Part B, below).

A. Regulation of Expression of tet Operator-Linked Nucleotide Sequences

Expression of a tet operator-linked nucleotide sequences is regulated bya transactivator fusion protein of the invention. Thus, the fusionprotein and the target nucleic acid are both present in a host cell ororganism. The presence of both the transactivator fusion protein and thetarget transcription unit in the same host cell or organism can beachieved in a number of different ways. For example, a host cell can betransfected with one nucleic acid of the expression system (e.g.,encoding the transactivator fusion protein), stably transfected cellscan be selected and then the transfected cells can be re-transfected(also referred to as "supertransfected") with nucleic acid correspondingto the other nucleic acid of the expression system (e.g., the targetnucleic acid to be transcribed). Two distinct selectable markers can beused for selection, e.g., uptake of the first nucleic acid can beselected with G418 and uptake of the second nucleic acid can be selectedwith hygromycin. Alternatively, a single population of cells can betransfected with nucleic acid corresponding to both components of thesystem. Accordingly, the invention provides a nucleic acid compositioncomprising:

a first nucleic acid encoding a fusion protein which activatestranscription, the fusion protein comprising a first polypeptide whichbinds to a tet operator sequence in the presence of tetracycline or atetracycline analogue operatively linked to a second polypeptide whichactivates transcription in eukaryotic cells; and

a second nucleic acid comprising a nucleotide sequence to be transcribedoperatively linked to at least one tet operator sequence.

In one embodiment, the two nucleic acids are two separate molecules(e.g., two different vectors). In this case, a host cell iscotransfected with the two nucleic acid molecules or successivelytransfected first with one nucleic acid molecule and then the othernucleic acid molecule. In another embodiment, the two nucleic acids arelinked (i.e., colinear) in the same molecule (e.g., a single vector). Inthis case, a host cell is transfected with the single nucleic acidmolecule.

The host cell may be a cell cultured in vitro or a cell present in vivo(e.g., a cell targeted for gene therapy). The host cell can further be afertilized oocyte, embryonic stem cell or any other embryonic cell usedin the creation of non-human transgenic or homologous recombinantanimals. Transgenic or homologous recombinant animals which compriseboth nucleic acid components of the expression system can be created byintroducing both nucleic acids into the same cells at an embryonicstage, or more preferably, an animal which carries one nucleic acidcomponent of the system in its genome is mated to an animal whichcarries the other nucleic acid component of the system in its genome.Offspring which have inherited both nucleic acid components can then beidentified by standard techniques.

B. Coordinate Regulation of Expression of Two Nucleotide Sequences

In addition to providing a system for the regulated expression of asingle transcribed nucleotide sequence, the invention further permitscoordinated regulation of the expression of two nucleotide sequencesoperatively linked to the same tet operator sequence(s). Accordingly,another aspect of the invention pertains to a novel tet-regulatedtranscription unit for coordinate regulation of two genes. In thistranscription unit, the same tet operator sequence(s) regulates theexpression of two operatively linked nucleotide sequences that aretranscribed in opposite directions from the common tet operatorsequence(s). Accordingly, one nucleotide sequence is operatively linkedto one side of the tet operator sequence (e.g., the 5' end on the topstrand of DNA) and the other nucleotide sequence is operatively linkedto the opposite side of the tet operator sequence (e.g., the 3' end onthe top strand of DNA). Additionally, it should be understood that eachnucleotide sequence to be transcribed includes an operatively linkedminimal promoter sequence which is located between the nucleotidesequence to be transcribed and the tet operator sequence(s).

A representative example of such a transcription unit is diagrammedschematically in FIG. 6. In this vectors, the two nucleotide sequences,operatively linked to the same tet operator sequence(s), are transcribedin opposite directions relative to the tet operator sequence(s) (i.e.,the sequences are transcribed in a divergent manner upon activation by atransactivator fusion protein of the invention). By "transcribed inopposite directions relative to the tet operator sequence(s)", it ismeant that the first nucleotide sequence is transcribed 5' to 3' fromone strand of the DNA (e.g., the bottom strand) and the secondnucleotide sequence is transcribed 5' to 3' from the other stand of theDNA (e.g., the top strand), resulting in bidirectional transcriptionaway from the tet operator sequence(s).

Accordingly, the invention provides a recombinant vector forcoordinately-regulated, bidirectional transcription of two nucleotidesequence. In one embodiment, the vector comprises a nucleotide sequencelinked by phosphodiester bonds comprising, in a 5' to 3' direction:

a first nucleotide sequence to be transcribed, operatively linked to

at least one tet operator sequence, operatively linked to

a second nucleotide sequence to be transcribed,

wherein transcription of the first and second nucleotide sequencesproceeds in opposite directions from the at least one tet operatorsequence(s) (i.e., the first and second nucleotide sequences aretranscribed in a divergent manner).

In another embodiment, the vector does not include the first and secondnucleotide sequence to be transcribed but instead contains cloning siteswhich allow for the introduction into the vector of nucleotide sequencesof interest. Accordingly, in this embodiment, the vector comprises anucleotide sequence comprising in a 5' to 3' direction:

a first cloning site for introduction of a first nucleotide sequence tobe transcribed, operatively linked to

at least one tet operator sequence, operatively linked to

a second cloning site for introduction of a second nucleotide sequenceto be transcribed,

wherein transcription of a first and second nucleotide sequenceintroduced into the vector proceeds in opposite directions from the atleast one tet operator sequence(s). It will be appreciated by thoseskilled in the art that this type of "cloning vector" may be in a formwhich also includes minimal promoter sequences such that a firstnucleotide sequence introduced into the first cloning site isoperatively linked to a first minimal promoter and a second nucleotidesequence introduced into the second cloning site is operatively linkedto a second minimal promoter. Alternatively, the "cloning vector" may bein a form which does not include minimal promoter sequences and instead,nucleotide sequences including linked minimal promoter sequences areintroduced into the cloning sites of the vector.

The term "cloning site" is intended to encompass at least onerestriction endonuclease site. Typically, multiple different restrictionendonuclease sites (e.g., a polylinker) are contained within the nucleicacid.

In yet another embodiment, the vector for coordinate, bidirectionaltranscription of two nucleotide sequences may contain a first nucleotideto be transcribed, such as that encoding a detectable marker (e.g.,luciferase or β-galactosidase), and a cloning site for introduction of asecond nucleotide sequence of interest.

The nucleotide sequences of two different suitable bidirectionalpromoter regions for use in a vector for coordinate regulation of twonucleotide sequences to be transcribed, as described herein, are shownin FIGS. 7A and 7B (SEQ ID NOS: 6 and 7, respectively). In the constructof FIG. 7A, both minimal promoters present in the construct are derivedfrom a CMV promoter. In the construct of FIG. 7B, one minimal promoterpresent in the construct is derived from a CMV promoter, whereas thesecond minimal promoter is derived from a TK promoter. A plasmidpUHDG1316-8, comprising a bidirectional promoter of the invention, hasbeen deposited on July 8, 1994 under the provisions of the BudapestTreaty at the Deutsche Sammlung Von Mikroorganismen und ZellKulturenGmbH (DSM) in Braunschweig, Germany and assigned deposit number DSM9281.

The transcription unit of the invention for bidirectional transcriptionof two nucleotide sequences operatively linked to the same tet operatorsequence(s) is useful for coordinating the expression of the twonucleotide sequences of interest. Preferably, at least one of thenucleotide sequences to be transcribed is a eukaryotic nucleotidesequence. In one application, the vector is used to producestoichiometric amounts of two subunits of a heterodimeric molecule inthe same cell. For example, the vector can be used produce antibodyheavy and light chains in the same cell or to produce growth factorreceptor subunits in the same cells. In another application, the vectoris used to express two gene products that cooperate in establishing aparticular cellular phenotype. In yet another application, the vector isused to coexpress an indicator function and a gene of interest, whereinthe indicator is utilized to monitor expression of the gene of interest.Thus, one of the two coordinately expressed sequences can encode a geneof interest and the other can encode a detectable marker, such as asurface marker or enzyme (e.g., β-galactosidase or luciferase) which isused for selection of cells expressing the gene of interest.

Transcription of the two coordinately-regulated nucleotide sequences canbe induced by tetracycline (or an analogue thereof) by use of theTc-inducible transcriptional activator of the invention to regulateexpression of the two nucleotide sequences. Thus, in this system,expression of both nucleotide sequences is "off" in the absence of Tc(or analogue), whereas expression is turned "on" by the presence of Tc(or analogue). Alternatively, the vector for coordinate regulation oftwo nucleotide sequences can be used in conjunction with othertetracycline-regulated transcription factors known in the art. Forexample, a transactivator fusion protein of a wild-type Tet repressorfused to a transcriptional activation domain, which activates geneexpression in the absence of Tc (or analogue), such as the tTA describedin Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA89:5547-5551, can also be used in conjunction with this targettranscription unit for coordinate regulation.

C. Independent Regulation of Expression of Multiple Nucleotide Sequences

The invention still further permits independent and opposite regulationof two or more nucleotide sequences to be transcribed. Accordingly,another aspect of the invention pertains to a novel tet-regulatedtranscription unit for independent regulation of two or more genes. Toindependently regulate the expression of two nucleotide sequences to betranscribed, one nucleotide sequence is operatively linked to a tetoperator sequence(s) of one class type and the other nucleotide sequenceis operatively linked to a tet operator sequence(s) of another classtype. Accordingly, the invention provides at least one recombinantvector for independent regulation of transcription of two nucleotidesequences. In one embodiment, the vector(s) comprises:

a first nucleotide sequence to be a transcribed operatively linked to atleast one tet operator sequence of a first class type; and

a second nucleotide sequence to be a transcribed operatively linked toat least one tet operator sequence of a second class type.

(It should be understood that each nucleotide sequence to be transcribedalso includes an operatively linked, upstream minimal promotersequence.) The two independently regulated transcription units can beincluded on a single vector, or alternatively, on two separate vectors.The recombinant vector(s) containing the nucleotide sequences to betranscribed can be introduced into a host cell or animal as describedpreviously.

In another embodiment, the vector(s) does not include the first andsecond nucleotide sequence to be transcribed but instead containscloning sites which allow for the introduction into the vector ofnucleotide sequences of interest. Accordingly, in this embodiment, thevector(s) comprises:

a first cloning site for introduction of a first nucleotide sequence tobe transcribed operatively linked to at least one tet operator sequenceof a first class type; and

a second cloning site for introduction of a second nucleotide sequenceto be transcribed operatively linked to at least one tet operatorsequence of a second class type.

This cloning vector(s) may be in a form that already includes first andsecond minimal promoters operatively linked, respectively, to the firstand second cloning sites. Alternatively, nucleotide sequences to betranscribed which include an operatively linked minimal promoter can beintroduced into the cloning vector.

In yet another embodiment, the vector for independent regulation of twonucleotide sequences may contain a first nucleotide to be transcribed,such as that encoding a detectable marker or a suicide gene, operativelylinked to at least one tet operator sequence of a first class type and acloning site for introduction of a second nucleotide sequence ofinterest such that it is operatively linked to at least one tet operatorsequence of a second class type.

It will be appreciated by those skilled in the art that variouscombinations of classes of tet operator sequences can be used forindependent regulation of two nucleotide sequences. For example, thefirst tet operator sequence(s) can be of the class A type and the secondcan be of the class B type, or the first tet operator sequence can be ofthe class B type and the second can be of the class C type, etc.Preferably, one to the two tet operators used is a class B typeoperator.

Independent transcription of the first and second nucleotide sequencesis regulated in a host cell by further introducing into the host cellone or more nucleic acids encoding two different transactivator fusionproteins which bind independently to tet operator sequences of differentclass types. The first fusion protein comprises a polypeptide whichbinds to a tet operator sequence in the presence of tetracycline or atetracycline analogue, operatively linked to a polypeptide whichactivates transcription in eukaryotic cells (e.g., a transactivatorfusion protein of the invention, such as a mutated Tn10-derived Tetrepressor linked to a VP16 activation region). The second fusion proteincomprises a polypeptide which binds to a tet operator sequence in theabsence of tetracycline or a tetracycline analogue, operatively linkedto a polypeptide which activates transcription in eukaryotic cells(e.g., a wild-type Tn10-derived Tet repressor linked to a VP16activation region, such as the tTA described in Gossen, M. and Bujard,H. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551). In one embodiment,the first fusion protein binds to the tet operator sequence of the firstclass type used in the transcription unit and the second fusion proteinbinds to the tet operator sequence of the second class type used in thetranscription unit. Alternatively, in another embodiment, the firstfusion protein binds to the second class type of tet operator and thesecond fusion protein binds to the first class type of tet operator.

For example, the first nucleotide sequence to be transcribed may belinked to a class A tet operator and the first fusion protein may bindto class A operators, whereas the second nucleotide sequence to betranscribed may be linked to a class B tet operator and the secondfusion protein may bind to class B operators. Thus, in this embodiment,transcription of the first nucleotide sequence is activated in thepresence of Tc (or analogue thereof) while transcription of the secondnucleotide sequence is activated in the absence of Tc (or analoguethereof). Alternatively, in another embodiment, the first fusion proteinbinds to class B operators and the second fusion protein binds to classA operators. In this case, transcription of the second nucleotidesequence is activated in the presence of Tc (or analogue thereof) whiletranscription of the first nucleotide sequence is activated in theabsence of Tc (or analogue thereof). Appropriate transactivator proteinsfor use in this system can be designed as described above in Section Iand in Gossen and Bujard (1992) cited supra. In order to inhibitheterodimerization between the two different types of Tet repressorfusion proteins present in the same cell, it may be necessary to mutatethe dimerization region of one or both of the transactivator fusionproteins. Mutations can be targeted to the C-terminal region of TetRknown to be involved in dimerization. The dimerization region has beendescribed in detail based upon the crystal structure of TetR (seeHinrichs, W. et al. (1994) Science 264:418-420).

This system allows for independent and opposite regulation of theexpression of two genes by Tc and analogues thereof. Use of different Tcanalogues as inducing agents may further allow for high, low orintermediate levels of expression of the different sequences (discussedin greater detail in Section V below). The novel transcription unit ofthe invention for independently regulating the expression of two genes,described above, can be used in situations where two gene products areto be expressed in the same cell but where it is desirable to expressone gene product while expression of the other gene product is turned"off", and vice versa. For example, this system is particularly usefulfor expressing in the same host cell either a therapeutic gene or asuicide gene (i.e., a gene which encodes a product that can be used todestroy the cell, such as ricin or herpes simplex virus thymidinekinase). In many gene therapy situations, it is desirable to be able toexpress a gene for therapeutic purposes in a host cell but also to havethe capacity to destroy the host cell once the therapy is completed.This can be accomplished using the above-described system by linking thetherapeutic gene to one class of tet operator and the suicide gene toanother class of tet operator. Thus, expression of the therapeutic genein a host cell can be stimulated by Tc (in which case expression of thesuicide gene is absent). Then, once the therapy is complete, Tc isremoved, which turns off expression of the therapeutic gene and turns onexpression of the suicide gene in the cell.

D. Combined Coordinate and Independent Regulation of Multiple NucleotideSequences

It is further possible to regulate the expression of four nucleotidesequences by combining the system described in Section IIIB with thesystem described in Section IIIC such that two pairs of sequences arecoordinately regulated while one pair is independently regulated fromthe other pair. Accordingly, two target transcription units can bedesigned comprising:

a first nucleic acid comprising in a 5' to 3' direction: a firstnucleotide sequence to be transcribed, a tet operator sequence(s) of afirst class type, and a second nucleotide sequence to be transcribed

a second nucleic acid comprising in a 5' to 3' direction: a thirdnucleotide sequence to be transcribed, a tet operator sequence(s) of asecond class type, and a fourth nucleotide sequence to be transcribed.

Transcription of the first and second nucleotide sequences in the firstnucleic acid proceeds in a divergent manner from the first class of tetoperator sequence(s). Likewise, transcription of the third and fourthnucleotide sequences in the second nucleic acid proceeds in a divergentmanner from the second class of tet operator sequence(s). Thus,expression of the first and second nucleotide sequences is coordinatelyregulated and expression of the third and fourth nucleotide sequences iscoordinately regulated. However, expression of the first and secondsequences is independently (and oppositely) regulated compared to thethird and fourth sequences through the use of two differenttransactivator fusion proteins, as described above, one which activatestranscription in the presence of Tc (or analogue thereof) and the otherwhich activates transcription in the absence of Tc (or analoguethereof). One transactivator is designed to bind to a tet operators ofthe first class type and the other is designed to bind to a tetoperators of the second class type. In other embodiments, rather thanalready containing first, second, third and/or fourth nucleotidesequences to be transcribed, these transcription units can containcloning sites which allow for the introduction of first, second, thirdand/or fourth nucleotide sequences to be transcribed.

IV. Tetracycline-Regulated Transcriptional Inhibitors

Another aspect of the invention pertains to transcriptional inhibitorfusion proteins. The inhibitor fusion proteins of the invention areconstructed similarly to the transactivator fusion proteins of theinvention (see Section I above) but instead of containing a polypeptidedomain that stimulates transcription in eukaryotic cells, the inhibitorfusion proteins contain a polypeptide domain that inhibits transcriptionin eukaryotic cells. The inhibitor fusion proteins are used todownregulate the expression of genes operably linked to tetO sequences.For example, when a tetO-linked gene is introduced into a host cell oranimal, the level of basal, constitutive expression of the gene may varydepending upon the type of cell or tissue in which the gene isintroduced and on the site of integration of the gene. Alternatively,constitutive expression of endogenous genes into which tetO sequenceshave been introduced may vary depending upon the strength of additionalendogenous regulatory sequences in the vicinity. The inhibitor fusionproteins described herein provide compositions that can be used toinhibit the expression of such tetO-linked genes in a controlled manner.

In one embodiment, the inhibitor fusion protein of the inventioncomprises a first polypeptide that binds to tet operator sequences inthe absence, but not the presence, of tetracycline (Tc) or an analoguethereof operatively linked to a heterologous second polypeptide thatinhibits transcription in eukaryotic cells. In another embodiment, theinhibitor fusion protein comprises a first polypeptide that binds to tetoperator sequences in the presence, but not the absence, of tetracyclineoperatively linked to a heterologous second polypeptide that inhibitstranscription in eukaryotic cells. The term "heterologous" is intendedto mean that the second polypeptide is derived from a different proteinthan the first polypeptide. Like the transactivator fusion proteins, thetranscriptional inhibitor fusion proteins can be prepared using standardrecombinant DNA techniques as described herein.

A. The first polypeptide of the transcriptional inhibitor fusion protein

The transcriptional inhibitor fusion protein of the invention iscomposed, in part, of a first polypeptide which binds to a tet operatorsequence either (i) in the absence, but not the presence of tetracycline(Tc), or an analogue thereof, or alternatively, (ii) in the presence,but not the absence of Tc or an analogue thereof.

Preferably, in the former embodiment, the first polypeptide is awild-type Tet repressor (which binds to tet operator sequences in theabsence but not the presence of Tc). A wild-type Tet repressor of anyclass (e.g., A, B, C, D or E) may be used as the first polypeptide.Preferably, the wild-type Tet repressor is a Tn10-derived Tet repressor.The nucleotide and amino acid sequences of a wild-type Tn10-derived Tetrepressor are shown in SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

Alternatively, in the latter embodiment, the first polypeptide is amutated Tet repressor as described in Section I, part A above (whichbinds to tet operator sequences in the presence but not the absence ofTc). A mutated Tet repressor of any class (e.g., A, B, C, D or E) may beused as the first polypeptide. Preferably, the mutated Tet repressor isa Tn10-derived Tet repressor having one or more amino acid substitutionsat positions 71, 95, 101 and/or 102. The nucleotide and amino acidsequences of such a mutated Tn10-derived Tet repressor are shown in SEQID NO: 18 and SEQ ID NO: 19, respectively.

B. The second polypeptide of the transcriptional inhibitor fusionprotein

The first polypeptide of the transcriptional inhibitor fusion protein isoperatively linked to a second polypeptide which directly or indirectlyinhibits transcription in eukaryotic cells. As described in Section I,above, to operatively link the first and second polypeptides of a fusionprotein, typically nucleotide sequences encoding the first and secondpolypeptides are ligated to each other in-frame to create a chimericgene encoding the fusion protein. However, the first and secondpolypeptides can be operatively linked by other means that preserve thefunction of each polypeptide (e.g., chemically crosslinked). Althoughthe fusion proteins are typically described herein as having the firstpolypeptide at the amino-terminal end of the fusion protein and thesecond polypeptide at the carboxy-terminal end of the fusion protein, itwill be appreciated by those skilled in the art that the oppositeorientation (i.e., the second polypeptide at the amino-terminal end andthe first polypeptide at the carboxy-terminal end) is also contemplatedby the invention.

Proteins and polypeptide domains within proteins which can function toinhibit transcription in eukaryotic cells have been described in the art(for reviews see, e.g., Renkawitz, R. (1990) Trends in Genetics6:192-197; and Herschbach, B. M. and Johnson, A. D. (1993) Annu. Rev.Cell. Biol. 9:479-509). Such transcriptional inhibitor domains have beenreferred to in the art as "silencing domains" or "repressor domains."Although the precise mechanism by which many of these polypeptidedomains inhibit transcription is not known (and the invention is notintended to be limited by mechanism), there are several possible meansby which repressor domains may inhibit transcription, including: 1)competitive inhibition of binding of either activator proteins or thegeneral transcriptional machinery, 2) prevention of the activity of aDNA bound activator and 3) negative interference with the assembly of afunctional preinitiation complex of the general transcription machinery.Thus, a repressor domain may have a direct inhibitory effect on thetranscriptional machinery or may inhibit transcription indirectly byinhibiting the activity of activator proteins. Accordingly, the term "apolypeptide that inhibits transcription in eukaryotic cells" as usedherein is intended to include polypeptides which act either directly orindirectly to inhibit transcription. As used herein, "inhibition" oftranscription is intended to mean a diminution in the level or amount oftranscription of a target gene compared to the level or amount oftranscription prior to regulation by the transcriptional inhibitorprotein. Transcriptional inhibition may be partial or complete. Theterms "silencer", "repressor" and "inhibitor" are used interchangeablyherein to describe a regulatory protein, or domains thereof, that caninhibit transcription.

A transcriptional "repressor" or "silencer" domain as described hereinis a polypeptide domain that retains its transcriptional repressorfunction when the domain is transferred to a heterologous protein.Proteins which have been demonstrated to have repressor domains that canfunction when transferred to a heterologous protein include the v-erbAoncogene product (Baniahmad, A. et al. (1992) EMBO J. 11:1015-1023), thethyroid hormone receptor (Baniahmad, supra), the retinoic acid receptor(Baniahmad, supra), and the Drosophila Krueppel (Kr) protein (Licht, J.D. et al. (1990) Nature 346:76-79; Sauer, F. and Jackle, H. (1991)Nature 3:563-566; Licht, J. D. et al. (1994) Mol. Cell. Biol.14:4057-4066). Non-limiting examples of other proteins which havetranscriptional repressor activity in eukaryotic cells include theDrosophila homeodomain protein even-skipped (eve), the S. cerevisiaeSsn6/Tup1 protein complex (see Herschbach and Johnson, supra), the yeastSIR1 protein (see Chien, et al. (1993) Cell 75:531-541), NeP1 (seeKohne, et al. (1993) J. Mol. Biol. 232:747-755), the Drosophila dorsalprotein (see Kirov, et al. (1994) Mol. Cell. Biol. 14:713-722; Jiang, etal. (1993) EMBO J. 12:3201-3209), TSF3 (see Chen, et al. (1993) Mol.Cell. Biol. 13:831-840), SF1 (see Targa, et al. (1992) Biochem. Biophys.Res. Comm. 188:416-423), the Drosophila hunchback protein (see Zhang, etal. (1992) Proc. Natl. Acad. Sci. USA 89:7511-7515), the Drosophilaknirps protein (see Gerwin, et al. (1994) Mol. Cell. Biol.14:7899-7908), the WT1 protein (Wilm's tumor gene product) (see Anant,et al. (1994) Oncogene 9:3113-3126; Madden et al., (1993) Oncogene8:1713-1720), Oct-2.1 (see Lillycrop, et al. (1994) Mol. Cell. Biol.14:7633-7642), the Drosophila engrailed protein (see Badiani, et al.(1994) Genes Dev. 8:770-782; Han and Manley, (1993) EMBO J.12:2723-2733), E4BP4 (see Cowell and Hurst, (1994) Nucleic Acids Res.22:59-65) and ZF5 (see Numoto, et al. (1993) Nucleic Acids Res.21:3767-3775).

In a preferred embodiment, the second polypeptide of the transcriptionalinhibitor fusion protein of the invention is a transcriptional silencerdomain of the Drosophila Krueppel protein. A C-terminal region havingrepressor activity can be used, such as amino acids 403-466 of thenative protein (see Sauer, F. and Jackle, H., supra). This region isreferred to as C64KR. The nucleotide and amino acid sequences of C64KRare shown in SEQ ID NO: 20 and SEQ ID NO: 21, respectively. Constructionof an expression vector encoding a TetR-C64KR fusion protein isdescribed in Example 4. Alternatively, an alanine-rich amino terminalregion of Kr that also has repressor activity can be used as the secondpolypeptide of the fusion protein. For example, amino acids 26-110 of Kr(see Licht, J. D. et al., (1990) supra) can be used as the secondpolypeptide. Alternatively, shorter or longer polypeptide fragmentsencompassing either of the Kr silencer domains that still retain full orpartial inhibitor activity are also contemplated (e.g., amino acids 62to 92 of the N-terminal silencer domain; see Licht, et al. (1994)supra).

In another preferred embodiment, the second polypeptide of thetranscriptional inhibitor fusion protein of the invention is atranscriptional silencer domain of the v-erbA oncogene product. Thesilencer domain of v-erbA has been mapped to approximately amino acidresidues 362-632 of the native v-erbA oncogene product (see Baniahmad,et al. supra). Accordingly, a fragment encompassing this region is usedas the second polypeptide of the silencer domain. In one embodiment,amino acid residues 364-635 of the native v-erbA protein are used. Thenucleotide and amino acid sequences of this region of v-erbA are shownin SEQ ID NO: 22 and SEQ ID NO: 23, respectively. Construction of anexpression vector encoding a TetR-v-erbA fusion protein is described inExample 5. Alternatively, shorter or longer polypeptide fragmentsencompassing the v-erbA silencer region that still retain full orpartial inhibitor activity are also contemplated. For example, a.a.residues 346-639, 362-639, 346-632, 346-616 and 362-616 of v-erbA may beused. Additionally, polypeptide fragments encompassing these regionsthat have internal deletions yet still retain fall or partial inhibitoractivity are encompassed by the invention, such as a.a. residues362-468/508-639 of v-erbA. Furthermore, two or more copies of thesilencer domain may be included in the fusion protein, such as twocopies of a.a. residues 362-616 of v-erbA. Suitable silencer polypeptidedomains of v-erbA are described further in Baniahmad, A. et al. (supra).

In other embodiments, other silencer domains are used. Non-limitingexamples of polypeptide domains that can be used include: amino acidresidues 120-410 of the thyroid hormone receptor alpha (THRα), aminoacid residues 143-403 of the retinoic acid receptor alpha (RARα), aminoacid residues 186-232 of knirps, the N-terminal region of WT 1 (seeAnant, supra), the N-terminal region of Oct-2.1 (see Lillycrop, supra),a 65 amino acid domain of E4BP4 (see Cowell and Hurst, supra) and theN-terminal zinc finger domain of ZF5 (see Numoto, supra). Moreover,shorter or longer polypeptide fragments encompassing these regions thatstill retain full or partial inhibitor activity are also contemplated.

In addition to previously described transcriptional inhibitor domains,novel transcriptional inhibitor domains, which can be identified bystandard techniques, are within the scope of the invention. Thetranscriptional inhibitor ability of a polypeptide can be assayed by: 1)constructing an expression vector that encodes the test silencerpolypeptide linked to another polypeptide having DNA binding activity(i.e., constructing a DNA binding domain-silencer domain fusionprotein), 2) cotransfecting this expression vector into host cellstogether with a reporter gene construct that is normally constitutivelyexpressed in the host cell and also contains binding sites for the DNAbinding domain and 3) determining the amount of transcription of thereporter gene construct that is inhibited by expression of the fusionprotein in the host cell. For example, a standard assay used in the artutilizes a fusion protein of a GAL4 DNA binding domain (e.g., amino acidresidues 1-147) and a test silencer domain. This fusion protein is thenused to inhibit expression of a reporter gene construct that containspositive regulatory sequences (that normally stimulate constitutivetranscription) and GAL4 binding sites (see e.g., Baniahmad, supra).

C. A third polypeptide of the transcriptional inhibitor fusion protein

In addition to a Tet repressor and a transcriptional silencer domain, atranscriptional inhibitor fusion protein of the invention can contain anoperatively linked third polypeptide which promotes transport of thefusion protein to a cell nucleus. As described for the transactivatorfusion proteins (see Section I, Part C, above), a nuclear localizationsignal can be incorporated into the transcriptional inhibitor fusionprotein.

D. Expression of the transcriptional inhibitor fusion protein

A nucleic acid molecule encoding a transcriptional inhibitor fusionprotein of the invention can be incorporated into a recombinantexpression vector and introduced into a host cell to express the fusionprotein in the host cell as described in Section II, Parts A, B and C,above. Preferably, a host cell expressing a transcriptional inhibitorfusion protein of the invention also carries a tet operator-linked geneof interest (i.e., target nucleotide sequence to be transcribed).

Transgenic organisms expressing a transcriptional inhibitor fusionprotein in cells thereof can be prepared as described in Section II,Part D, above. Moreover, homologous recombinant organisms expressing atranscriptional inhibitor fusion protein in cells thereof are alsoencompassed by the invention and can be prepared as described in SectionII, Part E, above. The invention provides recombinant expression vectorssuitable for homologous recombination. In one embodiment, such anexpression vector comprises a nucleic acid molecule encoding atranscriptional inhibitor fusion protein of the invention which isflanked at its 5' and 3' ends by additional nucleic acid of a eukaryoticgene, the additional nucleic acid being of sufficient length forsuccessful homologous recombination with the eukaryotic gene. Vectorsand methods for creating homologous recombinant organisms that expressthe components of the regulatory system of the invention, and usestherefor, are described in further detail in U.S. patent applicationSer. No. 08/260,452. Preferably, a transgenic or homologous recombinantorganism of the invention expressing a transcriptional inhibitor fusionprotein in cells thereof also carries a tet operator-linked gene ofinterest (i.e., target nucleotide sequence to be transcribed) in cellsthereof.

V. Kits of the Invention

Another aspect of the invention pertains to kits which include thecomponents of the inducible regulatory system of the invention. Such akit can be used to regulate the expression of a gene of interest (i.e.,a nucleotide sequence of interest to be transcribed) which can be clonedinto a target transcription unit. The kit may include nucleic acidencoding a transcriptional activator fusion protein or a transcriptionalinhibitor fusion protein or both. Alternatively, eukaryotic cells whichhave nucleic acid encoding a transactivator and/or inhibitor fusionprotein stably incorporated therein, such that the transactivator and/orinhibitor fusion protein are expressed in the eukaryotic cell, may beprovided in the kit.

In one embodiment, the kit includes a carrier means having in closeconfinement therein at least two container means: a first containermeans which contains a first nucleic acid (e.g., DNA) encoding atransactivator fusion protein of the invention (e.g., a recombinantexpression vector encoding a first polypeptide which binds to a tetoperator sequence in the presence of tetracycline operatively linked toa second polypeptide which activates transcription in eukaryotic cells),and a second container means which contains a second target nucleic acid(e.g., DNA) for the transactivator into which a nucleotide sequence ofinterest can be cloned. The second nucleic acid typically comprises acloning site for introduction of a nucleotide sequence to be transcribed(optionally including an operatively linked minimal promoter sequence)and at least one operatively linked tet operator sequence.

The term "cloning site" is intended to encompass at least onerestriction endonuclease site. Typically, multiple different restrictionendonuclease sites (e.g., a polylinker) are contained within the nucleicacid.

To regulate expression of a nucleotide sequence of interest using thecomponents of the kit, the nucleotide sequence is cloned into thecloning site of the target vector of the kit by conventional recombinantDNA techniques and then the first and second nucleic acids areintroduced into a host cell or animal. The transactivator fusion proteinexpressed in the host cell or animal then regulates transcription of thenucleotide sequence of interest in the presence of the inducing agent(Tc or analogue thereof).

Alternatively, in another embodiment, the kit includes a eukaryotic cellwhich is stably transfected with a nucleic acid encoding atransactivator fusion protein of the invention such that thetransactivator is expressed in the cell. Thus, rather than containingnucleic acid alone, the first container means described above cancontain a eukaryotic cell line into which the first nucleic acidencoding the transactivator has been stably introduced (e.g., by stabletransfection by a conventional method such as calcium phosphateprecipitation or electroporation, etc.). In this embodiment, anucleotide sequence of interest is cloned into the cloning site of thetarget vector of the kit and then the target vector is introduced intothe eukaryotic cell expressing the transactivator fusion protein.

Alternatively or additionally, a recombinant vector of the invention forcoordinate regulation of expression of two nucleotide sequences can alsobe incorporated into a kit of the invention. The vector can be includedin the kit in a form that allows for introduction into the vector of twonucleotide sequences of interest. Thus, in another embodiment, a kit ofthe invention includes 1) a first nucleic acid encoding a transactivatorfusion protein of the invention (or a eukaryotic cell into which thenucleic acid has been stably introduced) and 2) a second nucleic acidcomprising a nucleotide sequence comprising in a 5' to 3' direction: afirst cloning site for introduction of a first nucleotide sequence ofinterest operatively linked to at least one tet operator sequenceoperatively linked to a second cloning site for introduction of a secondnucleotide sequence of interest, wherein transcription of the first andsecond nucleotide sequences proceeds in opposite directions from the atleast one tet operator sequence. Optionally, the vector can includeoperatively linked minimal promoter sequences.

In another embodiment, the vector can be in a form that already containsone nucleotide sequence to be transcribed (e.g., encoding a detectablemarker such as luciferase, β-galactosidase or CAT) and a cloning sitefor introduction of a second nucleotide sequence of interest to betranscribed.

The transcription units and transactivators of the invention forindependent regulation of expression of two nucleotide sequences to betranscribed can also be incorporated into a kit of the invention. Thetarget transcription units can be in a form which allows forintroduction into the transcription units of nucleotide sequences ofinterest to be transcribed. Thus, in another embodiment, a kit of theinvention includes 1) a first nucleic acid encoding a transactivatorwhich binds to a tet operator of a first class type in the presence ofTc or an analogue thereof, 2) a second nucleic acid comprising a firstcloning site for introduction of a first nucleotide sequence to betranscribed operatively linked to at least one tet operator of a firstclass type, 3) a third nucleic acid encoding a transactivator whichbinds to a tet operator of a second class type in the absence of Tc oran analogue thereof, , and 4) a fourth nucleic acid comprising a secondcloning site for introduction of a second nucleotide sequence to betranscribed operatively linked to at least one tet operator of a secondclass type. (Optionally, minimal promoter sequences are included in thesecond and fourth nucleic acids). In another embodiment, one nucleotidesequence to be transcribed (e.g., encoding a suicide gene) is alreadycontained in either the second or the fourth nucleic acid. In yetanother embodiment, the nucleic acids encoding the transactivators(e.g., the first and third nucleic acids described above) can be stablyintroduced into a eukaryotic cell line which is provided in the kit.

In yet another embodiment, a kit of the invention includes a firstcontainer means containing a first nucleic acid encoding atranscriptional inhibitor fusion protein of the invention (e.g., thefusion protein inhibits transcription in eukaryotic cells either only inthe presence of Tc or only the absence of Tc) and a second containermeans containing a second nucleic acid comprising a cloning site forintroduction of a nucleotide sequence to be transcribed operativelylinked to at least one tet operator sequence. The kit may furtherinclude a third nucleic acid encoding a transactivator fusion proteinthat binds to tetO sequences either only in the presence of Tc or onlyin the absence of Tc. Alternatively, the first and/or third nucleicacids (i.e., encoding the inhibitor or transactivator fusion proteins)may be stably incorporated into a eukaryotic host cell which is providedin the kit.

In still another embodiment, a kit of the invention may include at leastone tetracycline or tetracycline analogue. For example, the kit mayinclude a container means which contains tetracycline,anhydrotetracycline, doxycycline, epioxytetracycline or othertetracycline analogue described herein.

VI. Regulation of Gene Expression by Tetracycline or Analogues Thereof

A. Stimulation of Gene Expression by Transactivator Fusion Proteins

In a host cell which carries nucleic acid encoding a transactivatorfusion protein of the invention and a nucleotide sequence operativelylinked to the tet operator sequence(i.e., gene of interest to betranscribed), high level transcription of the nucleotide sequenceoperatively linked to the tet operator sequence(s) does not occur in theabsence of the inducing agent, tetracycline or analogues thereof. Thelevel of basal transcription of the nucleotide sequence may varydepending upon the host cell and site of integration of the sequence,but is generally quite low or even undetectable in the absence of Tc. Inorder to induce transcription in a host cell, the host cell is contactedwith tetracycline or a tetracycline analogue. Accordingly, anotheraspect of the invention pertains to methods for stimulatingtranscription of a nucleotide sequence operatively linked to a tetoperator sequence in a host cell or animal which expresses atransactivator fusion protein of the invention. The methods involvecontacting the cell with tetracycline or a tetracycline analogue oradministering tetracycline or a tetracycline analogue to a subjectcontaining the cell.

The term "tetracycline analogue" is intended to include compounds whichare structurally related to tetracycline and which bind to the Tetrepressor with a K_(a) of at least about 10⁶ M⁻¹. Preferably, thetetracycline analogue binds with an affinity of about 10⁹ M⁻¹ orgreater. Examples of such tetracycline analogues include, but are notlimited to, anhydrotetracycline, doxycycline, chlorotetracycline,oxytetracycline and others disclosed by Hlavka and Boothe, "TheTetracyclines," in Handbook of Experimental Pharmacology 78, R. K.Blackwood et al. (eds.), Springer-Verlag, Berlin-New York, 1985; L. A.Mitscher, "The Chemistry of the Tetracycline Antibiotics", MedicinalResearch 9, Dekker, New York, 1978; Noyee Development Corporation,"Tetracycline Manufacturing Processes" Chemical Process Reviews, ParkRidge, N.J., 2 volumes, 1969; R. C. Evans, "The Technology of theTetracyclines", Biochemical Reference Series 1, Quadrangle Press, NewYork, 1968; and H. F. Dowling, "Tetracycline", Antibiotic Monographs,no. 3, Medical Encyclopedia, New York, 1955. Preferred Tc analogues forhigh level stimulation of transcription are anhydrotetracycline anddoxycycline. A Tc analogue can be chosen which has reduced antibioticactivity compared to Tc. Examples of such Tc analogues areanhydrotetracycline and epioxytetracycline.

To induce gene expression in a cell in vitro, the cell is contacted withTc or a Tc analogue by culturing the cell in a medium containing thecompound. When culturing cells in vitro in the presence of Tc or Tcanalogue, a preferred concentration range for the inducing agent isbetween about 10 and about 1000 ng/ml. Tc or a Tc analogue can bedirectly added to media in which cells are already being cultured, ormore preferably for high levels of gene induction, cells are harvestedfrom Tc-free media and cultured in fresh media containing Tc, or ananalogue thereof.

To induce gene expression in vivo, cells within in a subject arecontacted with Tc or a Tc analogue by administering the compound to thesubject. The term "subject" is intended to include humans and othernon-human mammals including monkeys, cows, goats, sheep, dogs, cats,rabbits, rats, mice, and transgenic and homologous recombinant speciesthereof. Furthermore, the term "subject" is intended to include plants,such as transgenic plants. When the inducing agent is administered to ahuman or animal subject, the dosage is adjusted to preferably achieve aserum concentration between about 0.05 and 1.0 μg/ml. Tc or a Tcanalogue can be administered to a subject by any means effective forachieving an in vivo concentration sufficient for gene induction.Examples of suitable modes of administration include oral administration(e.g., dissolving the inducing agent in the drinking water), slowrelease pellets and implantation of a diffusion pump. To administer Tcor a Tc analogue to a transgenic plant, the inducing agent can bedissolved in water administered to the plant.

The ability to use different Tc analogues as inducing agents in thissystem allows for modulate the level of expression of a tetoperator-linked nucleotide sequence. As demonstrated in Example 2,anhydrotetracycline and doxycycline have been found to be stronginducing agents. The increase in transcription of the target sequence istypically as high as 1000- to 2000-fold, and induction factors as highas 20,000 fold can be achieved. Tetracycline, chlorotetracycline andoxytetracycline have been found to be weaker inducing agents, i.e., theincrease in transcription of a target sequence is in the range of about10-fold. Thus, an appropriate tetracycline analogue is chosen as aninducing agent based upon the desired level of induction of geneexpression. It is also possible to change the level of gene expressionin a host cell or animal over time by changing the Tc analogue used asthe inducing agent. For example, there may be situations where it isdesirable to have a strong burst of gene expression initially and thenhave a sustained lower level of gene expression. Accordingly, ananalogue which stimulates a high levels of transcription can be usedinitially as the inducing agent and then the inducing agent can beswitched to an analogue which stimulates a lower level of transcription.Moreover, when regulating the expression of multiple nucleotidesequences (e.g., when one sequence is regulated by a one of class tetoperator sequence(s) and the other is regulated by another class of tetoperator sequence(s), as described above in Section III, Part C, above),it may be possible to independently vary the level of expression of eachsequence depending upon which transactivator fusion protein is used toregulate transcription and which Tc analogue(s) is used as the inducingagent. Different transactivator fusion proteins are likely to exhibitdifferent levels of responsiveness to Tc analogues. The level ofinduction of gene expression by a particular combination oftransactivator fusion protein and inducing agent (Tc or Tc analogue) canbe determined by techniques described herein, (e.g., see Example 2).Additionally, the level of gene expression can be modulated by varyingthe concentration of the inducing agent. Thus, the expression system ofthe invention provides a mechanism not only for turning gene expressionon or off, but also for "fine tuning" the level of gene expression atintermediate levels depending upon the type and concentration ofinducing agent used.

B. Inhibition of Gene Expression by Transcriptional Inhibitor FusionProteins

The invention also provides methods for inhibiting gene expression usingthe transcriptional inhibitor fusion proteins of the invention. Thesemethods can be used to down-regulate basal, constitutive ortissue-specific transcription of a tetO-linked gene of interest. Forexample, a gene of interest that is operatively linked to tetO sequencesand additional positive regulatory elements (e.g., consitutive ortissue-specific enhancer sequences) will be transcribed in host cells ata level that is primarily determined by the strength of the positiveregulatory elements in the host cell. Moreover, a gene of interest thatis operatively linked to tetOsequences and only a minimal promotersequence may exhibit varying degrees of basal level transcriptiondepending on the host cell or tissue and/or the site of integration ofthe sequence. In a host cell containing such a target sequence andexpressing an inhibitor fusion protein of the invention, transcriptionof the target sequence can be down regulated in a controlled manner byaltering the concentration of Tc (or analogue) in contact with the hostcell. For example, when the inhibitor fusion protein binds to tetO inthe absence of Tc, the concentration of Tc in contact with the host cellis reduced to inhibit expression of the target gene. Preferably, a hostcell is cultured in the absence of Tc to keep target gene expressionrepressed. Likewise, Tc is not administered to a host organism to keeptarget gene expression repressed. Alternatively, when the inhibitorfusion protein binds to tetO in the presence of Tc, the concentration ofTc in contact with the host cell is increased to inhibit expression ofthe target gene. For example, Tc is added to the culture medium of ahost cell or Tc is administered to a host organism to repress targetgene expression.

The inhibitor fusion proteins described herein can inhibit a tetO-linkedgene of interest in which the tetO sequences are positioned 5' of aminimal promoter sequence (e.g., tetracycline-regulated transcriptionunits as described in Section III, above). Furthermore, the inhibitorfusion protein may be used to inhibit expression of a gene of interestin which tetO-linked sequences are located 3' of the promoter sequencebut 5' of the transcription start site. Still further, the inhibitorfusion protein may be used to inhibit expression of a gene of interestin which tetO-linked sequences are located 3' of the transcription startsite.

Various Tc analogues as described in Section VI, part A, above, withrespect to the transactivator fusion proteins can similarly be used toregulate the activity of the inhibitor fusion proteins. Moreover, themethods of in vitro culture with Tc (or analogue) and in vivoadministration of Tc (or analogue) described in Section VI, part A, areequally applicable to the transcriptional inhibitor fusion proteins.

C Combined Positive and Negative Regulation of Gene Expression

In addition to regulating gene expression using either a transcriptionalactivator or inhibitor fusion protein alone, the two types of fusionproteins can be used in combination to allow for both positive andnegative regulation of expression of one or more target genes in a hostcell. Thus, a transcriptional inhibitor protein that binds to tetOeither (i) in the absence, but not the presence, of Tc, or (ii) in thepresence, but not the absence, of Tc, can be used in combination with atransactivator protein that binds to tetO either (i) in the absence, butnot the presence, of Tc, or (ii) in the presence, but not the absence,of Tc. Transactivator proteins that bind to tetO in the absence, but notthe presenc, of Tc (e.g., wild-type TetR-activator fusion proteins) aredescribed in further detail in U.S. Ser. No. 08/076,726, U.S. Ser. No.08/076,327 and U.S. Ser. No. 08/260,452. Transactivator fusion proteinsthat bind to tetO in the presence, but not the absence, of Tc (e.g.,mutated TetR-activator fusion proteins) are described herein (seeSection I above) and in U.S. Ser. No. 08/270,637 and U.S. Ser. No.08/275,876. Transcriptional inhibitor fusion proteins are describedherein in Section IV.

As described above in Section III, Part C, when more than one TetRfusion protein is expressed in a host cell or organism, additional stepsmay be taken to inhibit heterodimerization between the different TetRfusion proteins. For example, a transactivator composed of a TetR of oneclass may be used in combination with a transcriptional inhibitorcomposed of a TetR of a second, different class that does notheterodimerize with the first class of TetR. Alternatively, amino acidresidues of the TetR involved in dimerization may be mutated to inhibitheterodimerization. However, even if some heterodimerization betweentransactivator and inhibitor fusion proteins occurs in a host cell,sufficient amounts of homodimers should be produced to allow forefficient positive and negative regulation as described herein.

It will be appreciated by those skilled in the art that variouscombinations of activator and inhibitor proteins can be used to regulatea single tetO-linked gene of interest in both a positive and negativemanner or to regulate multiple tetO-linked genes of interest in acoordinated manner or in an independent manner using the teachingsdescribed herein. The precise regulatory components utilized will dependupon the genes to be regulated and the type of regulation desired.Several non-limiting examples of how the transactivator and inhibitorfusion proteins may be used in combination are described further below.However, many other possible combinations will be evident to the skilledartisan in view of the teachings herein and are intended to beencompassed by the invention.

In a preferred embodiment, illustrated schematically in FIG. 10,expression of a tetO-linked target gene of interest in a host cell isregulated in both a negative and positive manner by the combination ofan inhibitor fusion protein that binds to tetO in the absence, but notthe presence, of tetracycline or analogue thereof (referred to as atetracycline controlled silencing domain, or tSD) and an activatorfusion protein that binds to tetO in the presence, but not the absence,of tetracycline or analogue thereof (referred to as a reversetetracycline controlled transactivator, or rtTA). In addition to tetOsequences, the target gene is linked to a promoter, and may containother positive regulatory elements (e.g., enhancer sequences) thatcontribute to basal level, constitutive transcription of the gene in thehost cell. Binding of tSD to the tetO sequences in the absence oftetracycline or analogue (e.g., doxycycline) inhibits the basalconstitutive transcription of the gene of interest, thus keeping thegene of interest in a repressed state until gene expression is desired.When gene expression is desired, the concentration of tetracycline oranalogue (e.g., doxycycline) in contact with the host cell increased.Upon addition of the drug, tSD loses the ability to bind to tetOsequences whereas the previously unbound rtTA acquires the ability tobind to tetO sequences. The resultant binding of rtTA to the tetOsequences linked to the gene of interest thus stimulates transcriptionof the gene of interest. The level of expression may be controlled bythe concentration of tetracycline or analogue, the type of Tc analogueused, the duration of induction, etc., as described previously herein.It will be appreciated that the reverse combination of fusion proteins(i.e., the inhibitor binds in the presence but not the absence of thedrug and the activator binds in the absence but not the presence of thedrug) can also be used. In this case, expression of the gene of interestis kept repressed by contacting the host cell with the drug (e.g.,culture with Tc or analogue) and gene expression is activated by removalof the drug.

In another embodiment, the activator and inhibitor fusion proteins, asdescribed in the previous paragraph, are used in combination tocoordinately regulate, in both a positive and negative manner, two genesof interest using the bidirectional tetO-linked transcription unitdescribed in Section III, Part B above. In this case, Gene 1 and Gene 2are linked to the same tetO sequence(s), but in opposite orientations.The inhibitor fusion protein is used to repress basal levels oftranscription of both Gene 1 and Gene2 in a coordinate manner, whereasthe transactivator fusion protein is used to stimulate expression ofGene 1 and Gene 2 in a coordinate manner.

In yet another embodiment, the activator and inhbitor fusion proteinsare used to independently regulate two or more genes of interest usingthe tetO-linked transcription units as described in Section III, Part Cabove. For example, in one embodiment, a transactivator fusion proteinthat binds to one class of tetO sequences (e.g., class A) in thepresence, but not the absence of Tc or analogue is used in combinationwith an inhibitor fusion protein that binds to a second, different classof tetO sequences (e.g., class B) also in the presence, but not theabsence, of Tc or analogue. In a host cell containing Gene 1 linked toclass A tetO sequences and Gene 2 linked to class B tetO sequences, bothgenes will be expressed at basal levels in the absence of the drug,whereas expression of Gene 1 will be stimulated upon addition of thedrug and expression of Gene 2 will be repressed upon addition of thedrug.

Alternatively, in another embodiment, the transactivator binds to oneclass of tetO sequences (e.g., class A) in the presence, but not theabsence, of Tc or analogue and the inhibitor fusion protein binds to asecond, different class of tetO sequences (e.g., class B) in the absencebut not the presence of Tc or analogue. In the host cell as described inthe previous paragraph, Gene 1 will be expressed at basal levels in theabsence of the drug and will be stimulated upon addition of the drug,whereas Gene 2 will be repressed in the absence of the drug but willhave basal levels expression upon addition of the drug. Various otherpossible combinations will be apparent to the skilled artisan.Transactivator and inhibitor fusion proteins that bind to differentclasses of tetO sequences can be prepared as described in Section I,Part A. Target transcription units comprising tetO sequences ofdifferent classes can be prepared as described in Section III, Part C.

VII. Applications of the Invention

The invention is widely applicable to a variety of situations where itis desirable to be able to turn gene expression on and off, or regulatethe level of gene expression, in a rapid, efficient and controlledmanner without causing pleiotropic effects or cytotoxicity. Thus, thesystem of the invention has widespread applicability to the study ofcellular development and differentiation in eukaryotic cells, plants andanimals. For example, expression of oncogenes can be regulated in acontrolled manner in cells to study their function. Additionally, thesystem can be used to regulate the expression of site-specificrecombinases, such as CRE or FLP, to thereby allow for irreversiblemodification of the genotype of a transgenic organism under controlledconditions at a particular stage of development. For example, drugresistance markers inserted into the genome of transgenic plants thatallow for selection of a particular transgenic plant could beirreversibly removed via a Tc-regulated site specific recombinase. Otherapplications of the regulatory system of the invention include:

A. Gene Therapy

The invention may be particularly useful for gene therapy purposes, intreatments for either genetic or acquired diseases. The general approachof gene therapy involves the introduction of nucleic acid into cellssuch that one or more gene products encoded by the introduced geneticmaterial are produced in the cells to restore or enhance a functionalactivity. For reviews on gene therapy approaches see Anderson, W. F.(1992) Science 256:808-813; Miller, A. D. (1992) Nature 357:455-460;Friedmann, T. (1989) Science 244:1275-1281; and Cournoyer, D., et al.(1990) Curr. Opin. Biotech. 1:196-208. However, current gene therapyvectors typically utilize constitutive regulatory elements which areresponsive to endogenous transcriptions factors. These vector systems donot allow for the ability to modulate the level of gene expression in asubject. In contrast, the inducible regulatory system of the inventionprovides this ability.

To use the system of the invention for gene therapy purposes, in oneembodiment, cells of a subject in need of gene therapy are modified tocontain 1) nucleic acid encoding a transactivator fusion protein of theinvention in a form suitable for expression of the transactivator in thehost cells and 2) a gene of interest (e.g., for therapeutic purposes)operatively linked to a tet operator sequence(s). The cells of thesubject can be modified ex vivo and then introduced into the subject orthe cells can be directly modified in vivo (methods for modification ofthe cells are described above in Section II). Expression of the gene ofinterest in the cells of the subject is then stimulated by administeringTc or a Tc analogue to the patient. The level of gene expression can bevaried depending upon which particular Tc analogue is used as theinducing agent. The level of gene expression can also be modulated byadjusting the dose of the tetracycline, or analogue thereof,administered to the patient to thereby adjust the concentration achievedin the circulation and the tissues of interest.

Moreover, in another embodiment, a transcriptional inhibitor fusionprotein is used to further control the level of expression of the geneof interest. For example, the cells of the subject can be modified toalso contain a nucleic acid encoding a transcriptional inhibitor fusionprotein that binds to tetO in the absence of Tc. The nucleic acid is ina form suitable for expression of the inhibitor fusion protein in thehost cells. Thus, prior to administration of Tc (or analogue) to thesubject, the basal level of transcription of the gene of interest willbe kept silent by the inhibitor fusion protein. Upon administration ofTc, binding of the inhibitor fusion protein to tetO will be inhibitedwhereas binding of the transactivator fusion will be induced, therebystimulating transcription of the gene of interest. Such combinedpositive and negative regulation of gene expression using both atransactivator fusion protein and transcriptional inhibitor fusionprotein of the invention is illustrated schematically in FIG. 10.

Conventional detection methods known in the art, such as an enzymelinked immunosorbent assay, can be used to monitor the expression of theregulated protein of interest in the host cells and the concentration ofTc or Tc analogue can be varied until the desired level of expression ofthe protein of interest is achieved. Accordingly, expression of aprotein of interest can be adjusted according to the medical needs of anindividual, which may vary throughout the lifetime of the individual. Tostop expression of the gene of interest in cells of the subject,administration of the inducing agent is stopped. Thus, the regulatorysystem of the invention offers the advantage over constitutiveregulatory systems of allowing for modulation of the level of geneexpression depending upon the requirements of the therapeutic situation.

Genes of particular interest to be expressed in cells of a subject fortreatment of genetic or acquired diseases include those encodingadenosine deaminase, Factor VIII, Factor IX, dystrophin, β-globin, LDLreceptor, CFTR, insulin, erythropoietin, anti-angiogenesis factors,growth hormone, glucocerebrosidase, β-glucouronidase, α1-antitrypsin,phenylalanine hydroxylase, tyrosine hydroxylase, ornithinetranscarbamylase, arginosuccinate synthetase, UDP-glucuronysyltransferase, apoA1, TNF, soluble TNF receptor, interleukins (e.g.,IL-2), interferons (e.g., α- or γ-IFN) and other cytokines and growthfactors. Cells types which can be modified for gene therapy purposesinclude hematopoietic stem cells, myoblasts, hepatocytes, lymphocytes,skin epithelium and airway epithelium. For further descriptions of celltypes, genes and methods for gene therapy see e.g., Wilson, J. M et al.(1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano, D. et al.(1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Wolff, J. A. et al.(1990) Science 247:1465-1468; Chowdhury, J. R. et al. (1991) Science254:1802-1805; Ferry, N. et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Wilson, J. M. et al. (1992) J. Biol. Chem. 267:963-967;Quantin, B. et al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584; Dai,Y. et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; vanBeusechem, V. W. et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644;Rosenfeld, M. A. et al. (1992) Cell 68:143-155; Kay, M. A. et al. (1992)Human Gene Therapy 3:641-647; Cristiano, R. J. et al. (1993) Proc. Natl.Acad. Sci USA 90:2122-2126; Hwu, P. et al. (1993) J. Immunol.150:4104-4115; and Herz, J. and Gerard, R. D. (1993) Proc. Natl. Acad.Sci. USA 90:2812-2816.

Gene therapy applications of particular interest in cancer treatmentinclude overexpression of a cytokine gene (e.g., TNF-a) in tumorinfiltrating lymphocytes or ectopic expression of cytokines in tumorcells to induce an anti-tumor immune response at the tumor site),expression of an enzyme in tumor cells which can convert a non-toxicagent into a toxic agent, expression of tumor specific antigens toinduce an anti-tumor immune response, expression of tumor suppressorgenes (e.g., p53 or Rb) in tumor cells, expression of a multidrugresistance gene (e.g., MDR1 and/or MRP) in bone marrow cells to protectthem from the toxicity of chemotherapy.

Gene therapy applications of particular interest in treatment of viraldiseases include expression of trans-dominant negative viraltransactivation proteins, such as trans-dominant negative tat and revmutants for HIV or trans-dominant ICp4 mutants for HSV (see e.g.,Balboni, P. G. et al. (1993) J. Med. Virol. 41:289-295; Liem, S. E. etal. (1993) Hum. Gene Ther. 4:625-634; Malim, M. H. et al. (1992) J. Exp.Med. 176:1197-1201; Daly, T. J. et al. (1993) Biochemistry 32:8945-8954;and Smith, C. A. et al. (1992) Virology 191:581-588), expression oftrans-dominant negative envelope proteins, such as env mutants for HIV(see e.g., Steffy, K. R. et al. (1993) J. Virol. 67:1854-1859),intracellular expression of antibodies, or fragments thereof, directedto viral products ("internal immunization", see e.g., Marasco, W. A. etal. (1993) Proc. Natl. Acad. Sci. USA 90:7889-7893) and expression ofsoluble viral receptors, such as soluble CD4. Additionally, the systemof the invention can be used to conditionally express a suicide gene incells, thereby allowing for elimination of the cells after they haveserved an intended function. For example, cells used for vaccination canbe eliminated in a subject after an immune response has been generatedthe subject by inducing expression of a suicide gene in the cells byadministering Tc or a Tc analogue to the subject.

B. Production of proteins in Vitro

Large scale production of a protein of interest can be accomplishedusing cultured cells in vitro which have been modified to contain 1) anucleic acid encoding a transactivator fusion protein of the inventionin a form suitable for expression of the transactivator in the cells and2) a gene encoding the protein of interest operatively linked to a tetoperator sequence(s). For example, mammalian, yeast or fungal cells canbe modified to contain these nucleic acid components as describedherein. The modified mammalian, yeast or fungal cells can then becultured by standard fermentation techniques in the presence of Tc or ananalogue thereof to induce expression of the gene and produce theprotein of interest. Accordingly, the invention provides a productionprocess for isolating a protein of interest. In the process, a host cell(e.g., a yeast or fungus), into which has been introduced both a nucleicacid encoding a transactivator fusion protein of the invention and anucleic acid encoding the protein of the interest operatively linked toat least one tet operator sequence, is grown at production scale in aculture medium in the presence of tetracycline or a tetracyclineanalogue to stimulate transcription of the nucleotides sequence encodingthe protein of interest (i.e., the nucleotide sequence operativelylinked to the tet operator sequence(s)) and the protein of interest isisolated from harvested host cells or from the culture medium. Standardprotein purification techniques can be used to isolate the protein ofinterest from the medium or from the harvested cells.

C. Production of Proteins in Vivo

The invention also provides for large scale production of a protein ofinterest in animals, such as in transgenic farm animals. Advances intransgenic technology have made it possible to produce transgeniclivestock, such as cattle, goats, pigs and sheep (reviewed in Wall, R.J. et al. (1992) J. Cell. Biochem. 49:113-120; and Clark, A. J. et al.(1987) Trends in Biotechnology 5:20-24). Accordingly, transgeniclivestock carrying in their genome the components of the inducibleregulatory system of the invention can be constructed, wherein a geneencoding a protein of interest is operatively linked to at least one tetoperator sequence. Gene expression, and thus protein production, isinduced by administering Tc (or analogue thereof) to the transgenicanimal. Protein production can be targeted to a particular tissue bylinking the nucleic acid encoding the transactivator fusion protein toan appropriate tissue-specific regulatory element(s) which limitsexpression of the transactivator to certain cells. For example, amammary gland-specific regulatory element, such as the milk wheypromoter (U.S. Pat. No. 4,873,316 and European Application PublicationNo. 264,166), can be linked to the transactivator transgene to limitexpression of the transactivator to mammary tissue. Thus, in thepresence of Tc (or analogue), the protein of interest will be producedin the mammary tissue of the transgenic animal. The protein can bedesigned to be secreted into the milk of the transgenic animal, and ifdesired, the protein can then be isolated from the milk.

D. Animal Models of Human Disease

The transcriptional activator and inhibitor proteins of the inventioncan be used alone or in combination to stimulate or inhibit expressionof specific genes in animals to mimic the pathophysiology of humandisease to thereby create animal models of human disease. For example,in a host animal, a gene of interest thought to be involved in a diseasecan be placed under the transcriptional control of one or more tetoperator sequences (e.g., by homologous recombination, as describedherein). Such an animal can be mated to a second animal carrying one ormore transgenes for a transactivator fusion protein and/or an inhibitorfusion protein to create progeny that carry both atetracycline-regulated fusion protein(s) gene and a tet-regulated targetsequence. Expression of the gene of interest in these progeny can bemodulated using tetracycline (or analogue). For example, expression ofthe gene of interest can be downmodulated using a transcriptionalinhibitor fusion protein to examine the relationship between geneexpression and the disease. Such an approach may be advantageous overgene "knock out" by homologous recombination to create animal models ofdisease, since the tet-regulated system described herein allows forcontrol over both the levels of expression of the gene of interest andthe timing of when gene expression is down- or up-regulated.

E. Production of Stable Cell Lines for Gene Cloning and Other Uses

The transcriptional inhibitor system described herein can be used keepgene expression "off" (i.e., expressed) to thereby allow production ofstable cell lines that otherwise may not be produced. For example,stable cell lines carrying genes that are cytotoxic to the cells can bedifficult or impossible to create due to "leakiness" in the expressionof the toxic genes. By repressing gene expression of such toxic genesusing the transcriptional inhibitor fusion proteins of the invention,stable cell lines carrying toxic genes may be created. Such stable celllines can then be used to clone such toxic genes (e.g., inducing theexpression of the toxic genes under controlled conditions using Tc oranalog). General methods for expression cloning of genes, to which thetranscriptional inhibitor system of the invention can be applied, areknown in the art (see e.g., Edwards, C. P. and Aruffo, A. (1993) Curr.Opin. Biotech. 4:558-563) Moreover, the transcriptional inhibitor systemcan be applied to inhibit basal expression of genes in other cells tocreate stable cell lines, such as in embryonic stem (ES) cells. Residualexpression of certain genes introduced into ES stems may result in aninability to isolate stably transfected clones. Inhibition oftranscription of such genes using the transcriptional inhibitor systemdescribed herein may be useful in overcoming this problem.

Advantages

The inducible regulatory system of the invention utilizing atransactivator fusion protein addresses and overcomes many of thelimitations of other inducible regulatory systems in the art. Forexample, very high intracellular concentrations of the transcriptionalactivator fusion protein of the invention are not required for efficientregulation of gene expression. Additionally, since gene expression isinduced by adding rather than removing the inducing agent, the inductionkinetics in the system of the invention are not limited by the rate ofremoval of the inducing agent and thus are typically faster. Moreover,the inducing agent is only present when gene transcription is induced,thereby avoiding the need for the continuous presence of an agent tokeep gene expression off.

Use of the transcriptional inhibitor fusion proteins of the invention toinhibit transcription in eukaryotic cells also provide advantages overthe use of prokaryotic repressors alone (e.g., TetR, lacR) to inhibittranscription in eukaryotic cells. Since the inhibitor fusion proteinsof the invention contain a eukaryotic transcriptional silencer domain,these fusion proteins should be more efficient at repressingtranscription in eukaryotic cells, and thus may potentially requirelower intracellular concentrations for efficient repression with lessliklihood of "leakiness". Additionally, by insertion of tetO sequencesinto the regulatory region of an endogenous gene, the transcriptionalinhibitor fusion proteins of the invention can be used to down-regulateconstitutive and/or tissue-specific expression of endogenous genes.

Furthermore, in contrast to various versions of the lac system (e.g.,Labow et al. (1990) Mol. Cell. Biol. 10:3343-3356; Baim et al. (1991)Proc. Natl. Acad. Sci. USA 88:5072-5076), which are limited by thenegative properties of the inducing agent (IPTG) and/or by the need toincrease the temperature in order to induce gene expression (which mayelicit pleiotropic effects), the inducing agent used in the system ofthe invention (Tc or an analogue thereof) has many advantageousproperties: 1) Tc and analogues thereof exhibit high affinity for TetRand low toxicity for eukaryotic cells, and thus can be used for geneinduction at concentrations that do not affect cell growth ormorphology; 2) Tc analogues which retain TetR binding but which havereduced antibiotic activity exist and can be used as inducing agents,thereby avoiding possible side effects from the antibiotic property ofTc; 3) the pharmacokinetic properties of Tc and Tc analogues enablerapid and efficient cellular uptake and penetration of physiologicalbarriers, such as the placenta or the blood-brain barrier; and 4) Tcanalogues with different induction capabilities permit modulation of thelevel of gene expression.

Thus, the invention provides an inducible regulatory system which allowsfor rapid activation of gene transcription without cellular toxicity anda range of induction indices. The increase in gene expression uponinduction typically is between 1000- and 2000-fold and can be as high asabout 20,000-fold. Alternatively, lower levels of gene induction, e.g.,10-fold, can be achieved depending upon which inducing agent is used.This system can be utilized in a wide range of applications. Theseapplications include gene therapy, large-scale production of proteins incultured cells or in transgenic farm animals, and the study of genefunction, for example in relationship to cellular development anddifferentiation. Moreover, the novel transcription units of theinvention allow for coordinate or independent regulation of theexpression of multiple genes utilizing the regulatory components of theinvention.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are hereby incorporated by reference.

EXAMPLE 1 Selection of a Mutated Tet Repressor and Construction of aTetracycline Inducible Transcriptional Activator

A "reverse" Tet repressor, which binds to its target DNA in the presencerather than the absence of tetracycline, was generated by chemicalmutagenesis and selection essentially as described in Hecht, B. et al.(1993) J. Bacteriology 175:1206-1210. Single-stranded DNA (coding andnon-coding strands) encoding the wild-type Tn10-derived Tet repressorwas chemically mutagenized with sodium nitrite. Single-stranded DNAs (40μg in 40 μl in Tris-EDTA buffer) were mixed with 10 μl of 2.5M sodiumacetate (pH 4.3) and 50 μl of sodium nitrate ranging between 0.25M and2M and incubated for 45 to 60 minutes at room temperature. Aftermutagenesis, the complementary strand was synthesized using reversetranscriptase or by amplification using the polymerase chain reactionwith Taq DNA polymerase. Since the mutagenesis procedure yields multiplemutations in the DNA, three fragments of the gene, of about 200 basepairs each, were individually subcloned into a wt Tet repressor gene ina recombinant expression vector to replace the corresponding portion ofthe wild-type gene. This created a pool of mutated Tet repressor geneswherein each gene had mostly single mutations in the 200 base pairmutagenized fragment of the gene.

The pool of mutated Tet repressors were screened in a genetic assaywhich positively selects for a functional interaction between a Tetrepressor and its cognate operator using E. coli. strain WH207(λWH25)(the construction of this strain is described in detail in Wissmann, A.et al. (1991) Genetics 128:225-232). In this E. coli strain, tetoperators direct the expression of divergently arranged β-galactoside(lacZ) and Lac repressor (lacI) genes and the lac regulatory regiondirects the expression of a galactokinase (galK) gene. Binding of Tetrepressors to tet operators turns off transcription of the lacI and lacZgenes. The absence of Lac repressor allows for expression of the galKgene, which enables the E. coli strain to use galactose as a sole carbonsource, which serves as one marker. The lacZ⁻ phenotype serves as asecond marker. Thus, bacteria containing Tet repressors which bind totet operators have a Gal⁺, lacZ⁻ phenotype. Bacteria containingwild-type Tet repressors have a Gal⁺, lacZ⁻ phenotype in the absence oftetracycline. A mutated "reverse" Tet repressor (rTetR) was selectedbased upon a Gal⁺, lacZ⁻ phenotype in the presence of tetracycline.

The nucleotide and amino acid sequence of the rTetR mutant are shown inSEQ ID NOs: 1 (nucleotide positions 1-621) and 2 (amino acid positions1-207), respectively. Sequence analysis of the rTetR mutant showed thefollowing amino acid and nucleotide changes:

    ______________________________________                                        aa (position)          affected codon                                         wild-type mutant       wild-type                                                                              mutant                                        ______________________________________                                        glu(71)   lys          GAA      AAA                                           asp(95)   asn          GAT      AAT                                           leu(101)  ser          TTA      TCA                                           gly(102)  asp          GGT      GAT                                           ______________________________________                                    

Two additional mutations did not result in an amino acid exchange:

    ______________________________________                                        aa (position)          affected codon                                         wild-type mutant       wild-type                                                                              mutant                                        ______________________________________                                        leu(41)   leu          TTG      CTG                                           arg(80)   arg          CGT      CGC                                           ______________________________________                                    

To convert the rTetR mutant to a transcriptional activator, a 399 basepair XbaI/Eco47III fragment encoding amino acids 3 to 135 of rTetR(i.e., encompassing the mutated region) was exchanged for thecorresponding restriction fragment of the expression vector pUHD15-1 tocreate pUHD17-1. In pUHD15-1, nucleotide sequences encoding wild-typeTetR are linked in frame to nucleotide sequences encoding the C-terminal130 amino acids of herpes simplex virus VP16. These transactivatorsequences are flanked upstream by a CMV promoter/enhancer and downstreamby an SV40 poly(A) site (the construction of pUHD15-1 is described inmore detail in U.S. Ser. No. 08/076,726 and Gossen, M. and Bujard, H.(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551). Thus, in pUHD17-1,nucleotide sequences encoding the reverse TetR mutant are linked inframe to VP16 sequences to create a reverse Tc-controlled transactivator(referred to herein as tTA^(R)). The analogous exchange of the mutatedregion of rTetR for the wild-type region of TetR was performed withplasmid pUHD152-1, which is the same as pUHD15-1 except that itadditionally contains nucleotide sequences encoding a nuclearlocalization signal linked in-frame to the 5' end of the nucleotidesequences encoding the Tet repressor. The amino acid sequence of thenuclear localization signal is MPKRPRP (SEQ ID NO: 5), which is linkedto the serine at amino acid position 2 of TetR. The resulting expressionvector encoding the reverse Tc-controlled transactivator including anuclear localization signal (referred to herein as ntTA^(R)) was namedpUHD172-1.

EXAMPLE 2 Tetracycline-Induced Stimulation of Transcription by tTA^(R)Transient Transfection

The pUHD17-1 and pUHD172-1 expression vectors were transientlytransfected by a standard calcium phosphate method into HeLa cellstogether with a reporter plasmid, pUHC43-3, in which heptameric tetoperators are fused upstream of a minimal hCMV-promoter and a luciferasereporter gene (the reporter plasmid is described in detail in U.S. Ser.No. 08/076,726 and Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad.Sci. USA 89:5547-5551). After incubation of the transfected cells at 37°C. for 20 hours in the presence or absence of tetracycline (or ananalogue thereof), luciferase activity was assayed as follows: Cellsgrown to ˜80 % confluency in 35 mm dishes in Eagle's minimal essentialmedium were washed with 2 ml of phosphate-buffered saline before theywere lysed in 25 mM Tris phosphate, pH 7.8/2 mM dithiothreitol/2 mMdiaminocyclohexanetetraacetic acid/10% glycerol/1% Triton-X-100 for 10minutes at room temperature. The lysate was scraped off the culturedishes and centrifuged for 10 seconds in an Eppendorf centrifuge. Next,aliquots (10 μl) of the supernatant were mixed with 250 μl of 25 mMglycylglycine/15 mM MgSO4/5 mM ATP and assayed for luciferase activityin a Lumat LB9501 (Berthold, Wildbad, F.R.G.) using the integral mode(10 seconds). D-Luciferin (L6882, Sigma) was used at 0.5 mM. Thebackground signal measured in extracts of HeLa cells that did notcontain a luciferase gene was indistinguishable from the instrumentbackground (80-120 relative light units (rlu)/10 sec.). Protein contentof the lysate was determined according to Bradford (Bradford, M. M.(1976) Anal. Biochem. 72:248-254). Cells transfected with plasmidsencoding either tTA^(R) or ntTA^(R) showed an increased level ofluciferase activity in the presence of tetracyclines. This effect wasconsistently more pronounced when anhydrotetracycline (ATc) was usedinstead of tetracycline.

Stable Transfection

After this transient transfection analysis, expression vectors wereprepared for stable transfection of cells. A pSV2neo-derived neomycinresistance cassette (described in Southern, P. J. and Berg, P. (1982) J.Mol. Appl. Genet. 1:327-341) was integrated into the transactivatorexpression vectors pUHD17-1 and pUHD172-1, resulting in pUHD17-1 neo andpUHD172-1neo, respectively. pUHD172-1neo, coding for ntTA^(R), wasstably integrated into HeLa cells by standard techniques. TenG418-resistant cell clones were analyzed for their phenotype bytransient supertransfection with pUHC13-3 carrying the luciferase geneunder the control of a minimal CMV promoter and tet operators. Threeclones, HR4, HR5 and HR1O, showed a strong increase of luciferaseactivity in the presence of ATc. From these clones, HR5 was selected forfurther experiments.

To create stable transfectants for both ntTA^(R) and a tetoperator-linked luciferase reporter gene, HR5 cells were cotransfectedwith pUH13-3 and pHMR272, which encodes for hygromycin resistance (seeBernhard, H-U. et al. (1985) Exp. Cell Res. 158:237-243), and hygromycinresistant clones were selected. In an analogous experiment, HR5 cellswere cotransfected with pUH13-7 and pHMR272. pUH13-7 contains a minimalpromoter sequence spanning position +19 to -37 of the HSVtk promoteradjacent to the heptameric tetO sequences, rather than a minimal CMVpromoter. From 21 hygromycin resistant clones, 10 showed inducibleluciferase activity upon addition of Tc or doxycycline (Dc) to theculture medium. Clones containing the luciferase reporter gene linked toa minimal CMV promoter are referred to as HR5-C, whereas thosecontaining the luciferase reporter gene linked to a minimal tk promoterare referred to HR5-T.

Six of the HR5 clones stably transfected with a ntTA^(R) -dependentreporter unit and previously shown to be responsive to tetracyclineswere grown in parallel in the absence or presence of 1 μg/mldoxycycline. About 3×10⁴ cells were plated in each 35 mm dish (4 dishesfor each clone). After growth for 60 hours, cells were harvested and theluciferase activity of the extracts (in relative light units (rlu)/μgextracted protein) was determined. As shown in Table 1, the absoluteexpression levels of six clones demonstrate that activation ofluciferase gene expression over 3 orders of magnitude is achieved inseveral of the double stable cell lines containing the ntTA^(R)regulatory system.

It should be noted that even higher induction factors (e.g., as high asa 20,000-fold increase in expression) could be achieved if, instead ofsimply adding the inducing agent to the culture medium, the cells werewashed prior to induction and then replated in fresh culture mediumcontaining the inducing agent.

                  TABLE 1                                                         ______________________________________                                        Doxycycline-dependent luciferase activity                                     of double stable luc+/HR5 cell clones                                         Luciferase Activity, rlu/μg protein                                        Clone  -Doxycycline +Doxycycline                                                                             Induction Factor                               ______________________________________                                        HR5-C6 65           54,911      845                                                  62           69,525     1120                                           HR5-C11                                                                              100          165,671    1660                                                  142          179,651    1270                                           HR5-C14                                                                              43           44,493     1030                                                  43           56,274     1310                                           HR5-T2 56           16,696      298                                                  40           16,416      410                                           HR5-T15                                                                              6.8            1838      270                                                  6.5            1688      260                                           HR5-T19                                                                              4.8            1135      236                                                  5.4            1285      237                                           ______________________________________                                    

Induction of luciferase activity by different tetracyclines

The ability of tetracycline and several different tetracycline analoguesto induce luciferase expression in HR5-C11 cells was examined. HR5-C11cells plated at a density of about 3×10⁴ cells/35 mm dish (˜80%confluency). After full attachment of the cells, the followingtetracyclines ere added to the cultures at a concentration of 1 μg/ml:tetracycline-HCl (Tc), oxytetracycline-HCl (OTc), chlorotetracycline(CTc), anhydrotetracycline-HCl (ATc) and doxycycline-HCl (Doxy). Thesecompounds are commercially available from Sigma Chemical Co., St. Louis,Mo., and were kept in aqueous solution at a concentration of 1 μg/ml.Cells grown in the absence of antibiotic (-) served as a control. After3 days, the cells were harvested and the luciferase activity and theprotein content of the extracts were determined. The results are shownin the bar graph of FIG. 1. Each bar in the figure (closed and hatched)represents the relative luciferase activity (normalized toward theamount of extracted protein) of a single culture dish. The mean of theluciferase activities obtained from the two plates grown withouttetracyclines was defined as 1. Tc, CTc and OTc showed modeststimulation of luciferase activity. By contrast, ATc and Doxy stimulatedluciferase activity approximately 1000 and 1500 fold, respectively.

Dose-response of luciferase activity to doxycycline in HR5-C11 cells

The above-described experiment examining the induction ability ofdifferent tetracyclines revealed that doxycycline was the most potenteffector of the tetracyclines examined. Doxycycline was thereforeselected to quantitatively analyze its dose-response. HR5-C11 cells wereincubated with different concentrations of doxycycline and luciferaseactivity was measured. The data of three independent experiments areshown in FIG. 2. At less than 10 ng/ml in the culture medium,doxycycline is ineffective at inducing luciferase activity. However,when the concentration was raised above 10 ng/ml, an almost linearincrease in expression of luciferase was observed. Maximal activationwas achieved at 1 μg/ml. At concentrations above 3 μg/ml, doxycyclineshowed a slight growth-inhibitory effect on HeLa cells as determined ina MTT-assay.

Kinetics of induction of the ntTA^(R) system

To examine the kinetics of doxycycline-induced ntTA^(R) -mediatedinduction of gene expression, the time course of induction of luciferaseactivity in HR5-C11 cells was monitored after addition of doxycycline tothe medium (final concentration 1 μg/ml). Cells were cultured in thepresence of doxycycline and after various time intervals, the cells wereharvested and luciferase activity was determined as described above. Asshown in FIG. 3, a 100-fold induction of luciferase activity wasobserved after 5.5 hours incubation with Doxy. Fully induced levels wereachieved in less than 24 hours of incubation with Doxy. Thus, theseresults indicate that induction of gene expression occurs rapidlyfollowing exposure of the cells to the inducing agent.

EXAMPLE 3 Coordinate Regulation of the Expression of Two NucleotideSequences by a Tc-Controlled Transcriptional Activator

A recombinant expression vector for coordinate, bidirectionaltranscription of two nucleotide sequences was constructed comprising, ina 5' to 3' direction: a luciferase gene, a first minimal promoter, seventet operator sequences, a second minimal promoter and a LacZ gene. Theconstruct is illustrated in FIG. 6. In this construct, the luciferaseand LacZ genes are oriented such that they are transcribed in oppositeorientations relative to the tet operator sequences, i.e., theluciferase gene is transcribed in a 5' to 3' direction from the bottomstrand of DNA, whereas the LacZ gene is transcribed in a 5' to 3'direction from the top strand of DNA. The luciferase gene is followed byan SV40 polyadenylation signal, whereas the LacZ gene is followed by aβ-globin polyadenylation signal.

The construct was transfected into the HeLa cell line HtTA-1 cells,which express a wild-type Tet repressor-VP16 fusion protein (referred toas tTA and described in Gossen, M. and Bujard, H. (1992) Proc. Natl.Acad. Sci. USA 89:5547-5551). The tTA fusion protein binds to tetoperator sequences in the absence of Tc (or analogue) but not in thepresence of Tc (or analogue). The construct was cotransfected intoHtTA-1 cells with a plasmid which confers hygromycin resistance andstably transfected clones were selected based upon their hygromycinresistant phenotype. Selected hygromycin resistant (Hygr^(r)) cloneswere examined for luciferase and β-galactosidase activity. Clonespositive for all three markers (Hygr^(r), luc⁺, β-gal⁺) were thenexamined for tetracycline-dependent coregulation of expression ofluciferase and β-galactosidase activity by culturing the clones inincreasing amounts of tetracycline and measuring luciferase andβ-galactosidase activity. The results of such an experiment using cloneHt1316-8/50 are shown in FIG. 8. In the absence of tetracycline (inwhich case tTA can bind to tet operators and activate gene expression),both luciferase and β-galactosidase activity is detected. In thepresence of increasing amounts of tetracycline, luciferase andβ-galactosidase activity are coordinately and equivalentlydownregulated. This data demonstrates that expression of two genes canbe coordinately regulated by a tetracycline-controlled transactivator byoperatively linking the two genes to the same tet operator sequence(s).

EXAMPLE 4 Construction of a Tetracycline-Regulated TranscriptionalInhibitor Fusion Protein Comprising TetR and a Krueppel Silencer Domain

To contruct an expression vector encoding a tetracycline-regulatedtranscriptional inhibitor of the invention (also referred to as atetacycline controlled silencer domain, or tSD), a nucleic acid fragmentencoding a transcriptional silencer domain is ligated into an expressionvector containing nucleotide sequences encoding a wild-type or modified(i.e., mutated) TetR such that the silencer domain coding sequences areligated in-frame with the TetR coding sequences. The plasmidpUHD141sma-1 contains nucleotide sequences encoding a wild-typeTn10-derived Tet repressor (the nucleotide and amino acid sequences ofwhich are shown in SEQ ID NOs: 16 and 17, respectively). InpUHD141sma-1, the TetR coding sequence is linked at its 5' end to a CMVpromoter and at its immediate 3' end to a nucleotide sequence thatcreates a polylinker into which additional nucleic acid fragments can beintroduced. The nucleotide sequence across this polylinker region is:TCC CCG GGT AAC TAA GTA AGG ATC C (SEQ ID NO: 24) (wherein TCC CCG GGTACC encode amino acid residues 205-208 of TetR, namely Ser-Gly-Ser-Asn).This polylinker region includes restriction endonuclease sites for PspAI(CCC GGG) and BamHi (GGA TCC). Downstream of the polylinker region, theplasmid contains an SV40-derived polyadenylation signal. The pUHD141sma-1 vector is illustrated schematically in FIG. 11.

To construct an expression vector encoding a fusion protein between TetRand a transcriptional silencer domain from the Drosophila Krueppel (Kr)protein, a nucleic acid fragment encoding a silencer domain from Kr isamplified by the polymerase chain reaction (PCR) using Kr cDNA as atemplate. Oligonucleotide primers are designed which amplify a nucleicacid fragment encoding the C-terminal 64 amino acids of Kr (referred toas C64KR). This region corresponds to amino acid positions 403-466 ofthe native protein. The nucleotide and amino acid sequences of C64KR areshown in SEQ ID NO: 20 and SEQ ID NO: 21, respectively. PCR primers aredesigned to include restriction endonuclease sites such that theresultant amplified fragment contains restriction endonuclease sites atits 5' and 3' ends. Restriction endonuclease sites are chosen that arecontained within the polylinker of pUHD141sma-1 which allow in-frame,directional ligation of the amplified fragment into the polylinker site.For example, PCR primers are designed which incorporate a PspAI site(CCC GGG) at the 5' end of the fragment encoding C64KR and a BamHI siteat the 3' end of the fragment. After a standard PCR reaction, theamplified fragment and pUHD141-sma1 are digested with PspAI and BamHI.The amplified fragment is then ligated directionally into the polylinkersite of pUHD141-sma1 using standard ligation conditions to create theexpression vector pUHD141kr-1. Standard techniques are used to isolatethe desired plasmid and confirm its construction. Construction ofpUHD141kr-1 is illustrated schematically in FIG. 11.

The resultant pUHD 14 1kr-1 expression vector contains nucleotidesequences encoding a fusion protein comprising amino acids 1-207 of thewild type TetR linked in-frame to amino acids 403-466 of Kr (C64KR). Thenucleotide and amino acid sequences across the junction of the fusionprotein are as follows: AGT GGG TCC CCG GGT GAC ATG GAA (SEQ ID NO: 25)and Ser-Gly-Ser-Pro-Gly-Asp-Met-Glu (SEQ ID NO: 26). Ser-Gly-Sercorresponds to amino acids 205-207 of TetR, Pro-Gly are encoded by thepolylinker and Asp-Met-Glu correspond to the amino acids 403-405 ofC64KR.

Similarly, an expression vector encoding a fusion protein of a mutatedTetR (that binds to tetO only in the presence of Tc) and C64KR can beconstructed as described above using nucleotide sequences encoding amutant TetR (the nucleotide and amino acid sequences of which are shownin SEQ ID NOs: 18 and 19, respectively) in place of the wild type TetRsequences in pUHD141 -sma1.

An expression vector encoding a TetR-Kr fusion protein (e.g.,pUHD141kr-1) is transiently or stably transfected into host cells asdescribed in Example 2 to express the TetR-Kr fusion protein in the hostcell. A reporter gene construct containing one or more tetO sequences, aminimal promoter and a reporter gene, such as luciferase, is alsotransfected into the cells as described in Example 2. (Reporter geneconstructs are described in further detail in U.S. Ser. No. 08/076,726and Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA89:5547-5551). Luciferase activity in the presence and absence ofincreasing concentrations of Tc or an analogue, e.g., doxycycline, ismeasured as described in Example 2. For the wild type TetR-Kr fusionprotein described above, the transcriptional inhibiting ability of thefusion protein is determined by comparing the amount of luciferaseactivity in the presence of doxycycline (no repression) to the amount ofluciferase activity in the absence of doxycycline (repression). Thetranscriptional inhibiting activity of the fusion protein can also betested using reporter gene constructs that exhibit higher basal levelsof expression (i.e., higher levels of expression in the presence ofdoxycycline) by using a reporter gene construct that contains additionalpositive regulatory elements (e.g., enhancer sequences).

EXAMPLE 5 Construction of a Tetracycline-Regulated TranscriptionalInhibitor Fusion Protein Comprising TetR and a v-erbA Silencer Domain

To construct an expression vector encoding a fusion protein between TetRand a transcriptional silencer domain from the v-erbA oncogene product,a nucleic acid fragment encoding a silencer domain from v-erbA isligated in-frame into pUHD141sma-1 as described in Example 4. A nucleicacid fragment encoding a v-erbA silencer domain suitable for ligationinto pUHD141sma-1 is amplified by the polymerase chain reaction (PCR)using a v-erbA cDNA as a template. Oligonucleotide primers are designedwhich amplify a nucleic acid fragment encoding amino acids 364-635 ofthe native v-erbA protein. The nucleotide and amino acid sequences ofthis region of v-erbA are shown in SEQ ID NO: 22 and SEQ ID NO: 23,respectively. As described in Example 4, PCR primers are designed suchthat the amplified v-erbA fragment contains restriction endonucleasesites at its 5' and 3' ends, such as PspAI at the 5' end and BamHI atthe 3' end. After a standard PCR reaction, the amplified fragment andpUHD141-sma1 are digested with PspAI and BamHI. The amplified fragmentis then ligated directionally into the polylinker site of pUHD141-sma1using standard ligation conditions to create the expression vectorpUHD141kr-1. Standard techniques can be used to isolate the desiredplasmid and confirm its construction. Construction of pUHD141erb-1 isillustrated schematically in FIG. 11.

The resultant pUHD141 erb-1 expression vector contains nucleotidesequences encoding a fusion protein comprising a wild type TetR linkedin-frame to amino acids 364-635 of v-erbA. The nucleotide and amino acidsequences across the junction of the fusion protein are as follows: AGTGGG TCC CCG GGT CTG GAC GAC (SEQ ID NO: 27) andSer-Gly-Ser-Pro-Gly-Leu-Asp-Asp (SEQ ID NO: 28). Ser-Gly-Ser correspondsto amino acids 205-207 of TetR, Pro-Gly are encoded by the polylinkerand Leu-Asp-Asp correspond to amino acids 364-366 of the v-erbA silencerdomain.

As described in Example 4, an expression vector encoding a fusionprotein of a mutated TetR (that binds to tetO only in the presence ofTc) and a v-erbA silencer domain can be constructed as described aboveusing nucleotide sequences encoding a mutant TetR (the nucleotide andamino acid sequences of which are shown in SEQ ID NOs: 18 and 19,respectively) in place of the wild type TetR sequences in pUHD141-sma1.

Expression of the TetR-v-erbA fusion protein in host cells and assayingof the transcriptional inhibiting activity of the fusion protein is asdescribed in Example 4 for the TetR-Kr fusion protein.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 28                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1008 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 1..1008                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mRNA                                                            (B) LOCATION: 1..1008                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: misc. binding                                                   (B) LOCATION: 1..207                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: misc. binding                                                   (B) LOCATION: 208..335                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1005                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTG48                            MetSerArgLeuAspLysSerLysValIleAsnSerAlaLeuGluLeu                              151015                                                                        CTTAATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAG96                            LeuAsnGluValGlyIleGluGlyLeuThrThrArgLysLeuAlaGln                              202530                                                                        AAGCTAGGTGTAGAGCAGCCTACACTGTATTGGCATGTAAAAAATAAG144                           LysLeuGlyValGluGlnProThrLeuTyrTrpHisValLysAsnLys                              354045                                                                        CGGGCTTTGCTCGACGCCTTAGCCATTGAGATGTTAGATAGGCACCAT192                           ArgAlaLeuLeuAspAlaLeuAlaIleGluMetLeuAspArgHisHis                              505560                                                                        ACTCACTTTTGCCCTTTAAAAGGGGAAAGCTGGCAAGATTTTTTACGC240                           ThrHisPheCysProLeuLysGlyGluSerTrpGlnAspPheLeuArg                              65707580                                                                      AATAAGGCTAAAAGTTTTAGATGTGCTTTACTAAGTCATCGCAATGGA288                           AsnLysAlaLysSerPheArgCysAlaLeuLeuSerHisArgAsnGly                              859095                                                                        GCAAAAGTACATTCAGATACACGGCCTACAGAAAAACAGTATGAAACT336                           AlaLysValHisSerAspThrArgProThrGluLysGlnTyrGluThr                              100105110                                                                     CTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAG384                           LeuGluAsnGlnLeuAlaPheLeuCysGlnGlnGlyPheSerLeuGlu                              115120125                                                                     AATGCATTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGC432                           AsnAlaLeuTyrAlaLeuSerAlaValGlyHisPheThrLeuGlyCys                              130135140                                                                     GTATTGGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACA480                           ValLeuGluAspGlnGluHisGlnValAlaLysGluGluArgGluThr                              145150155160                                                                  CCTACTACTGATAGTATGCCGCCATTATTACGACAAGCTATCGAATTA528                           ProThrThrAspSerMetProProLeuLeuArgGlnAlaIleGluLeu                              165170175                                                                     TTTGATCACCAAGGTGCAGAGCCAGCCTTCTTATTCGGCCTTGAATTG576                           PheAspHisGlnGlyAlaGluProAlaPheLeuPheGlyLeuGluLeu                              180185190                                                                     ATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTGGGTCCGCG624                           IleIleCysGlyLeuGluLysGlnLeuLysCysGluSerGlySerAla                              195200205                                                                     TACAGCCGCGCGCGTACGAAAAACAATTACGGGTCTACCATCGAGGGC672                           TyrSerArgAlaArgThrLysAsnAsnTyrGlySerThrIleGluGly                              210215220                                                                     CTGCTCGATCTCCCGGACGACGACGCCCCCGAAGAGGCGGGGCTGGCG720                           LeuLeuAspLeuProAspAspAspAlaProGluGluAlaGlyLeuAla                              225230235240                                                                  GCTCCGCGCCTGTCCTTTCTCCCCGCGGGACACACGCGCAGACTGTCG768                           AlaProArgLeuSerPheLeuProAlaGlyHisThrArgArgLeuSer                              245250255                                                                     ACGGCCCCCCCGACCGATGTCAGCCTGGGGGACGAGCTCCACTTAGAC816                           ThrAlaProProThrAspValSerLeuGlyAspGluLeuHisLeuAsp                              260265270                                                                     GGCGAGGACGTGGCGATGGCGCATGCCGACGCGCTAGACGATTTCGAT864                           GlyGluAspValAlaMetAlaHisAlaAspAlaLeuAspAspPheAsp                              275280285                                                                     CTGGACATGTTGGGGGACGGGGATTCCCCGGGTCCGGGATTTACCCCC912                           LeuAspMetLeuGlyAspGlyAspSerProGlyProGlyPheThrPro                              290295300                                                                     CACGACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTTCGAGTTT960                           HisAspSerAlaProTyrGlyAlaLeuAspMetAlaAspPheGluPhe                              305310315320                                                                  GAGCAGATGTTTACCGATCCCCTTGGAATTGACGAGTACGGTGGGTAG1008                          GluGlnMetPheThrAspProLeuGlyIleAspGluTyrGlyGly                                 325330335                                                                     (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 335 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetSerArgLeuAspLysSerLysValIleAsnSerAlaLeuGluLeu                              151015                                                                        LeuAsnGluValGlyIleGluGlyLeuThrThrArgLysLeuAlaGln                              202530                                                                        LysLeuGlyValGluGlnProThrLeuTyrTrpHisValLysAsnLys                              354045                                                                        ArgAlaLeuLeuAspAlaLeuAlaIleGluMetLeuAspArgHisHis                              505560                                                                        ThrHisPheCysProLeuLysGlyGluSerTrpGlnAspPheLeuArg                              65707580                                                                      AsnLysAlaLysSerPheArgCysAlaLeuLeuSerHisArgAsnGly                              859095                                                                        AlaLysValHisSerAspThrArgProThrGluLysGlnTyrGluThr                              100105110                                                                     LeuGluAsnGlnLeuAlaPheLeuCysGlnGlnGlyPheSerLeuGlu                              115120125                                                                     AsnAlaLeuTyrAlaLeuSerAlaValGlyHisPheThrLeuGlyCys                              130135140                                                                     ValLeuGluAspGlnGluHisGlnValAlaLysGluGluArgGluThr                              145150155160                                                                  ProThrThrAspSerMetProProLeuLeuArgGlnAlaIleGluLeu                              165170175                                                                     PheAspHisGlnGlyAlaGluProAlaPheLeuPheGlyLeuGluLeu                              180185190                                                                     IleIleCysGlyLeuGluLysGlnLeuLysCysGluSerGlySerAla                              195200205                                                                     TyrSerArgAlaArgThrLysAsnAsnTyrGlySerThrIleGluGly                              210215220                                                                     LeuLeuAspLeuProAspAspAspAlaProGluGluAlaGlyLeuAla                              225230235240                                                                  AlaProArgLeuSerPheLeuProAlaGlyHisThrArgArgLeuSer                              245250255                                                                     ThrAlaProProThrAspValSerLeuGlyAspGluLeuHisLeuAsp                              260265270                                                                     GlyGluAspValAlaMetAlaHisAlaAspAlaLeuAspAspPheAsp                              275280285                                                                     LeuAspMetLeuGlyAspGlyAspSerProGlyProGlyPheThrPro                              290295300                                                                     HisAspSerAlaProTyrGlyAlaLeuAspMetAlaAspPheGluPhe                              305310315320                                                                  GluGlnMetPheThrAspProLeuGlyIleAspGluTyrGlyGly                                 325330335                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GACGCGCTAGACGATTTCGATCTGGACATGTTG33                                           AspAlaLeuAspAspPheAspLeuAspMetLeu                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AspAlaLeuAspAspPheAspLeuAspMetLeu                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       MetProLysArgProArgPro                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 569 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GAATTCGGGGCCGCGGAGGCTGGATCGGTCCCGGTGTCTTCTATGGAGGTCAAAACAGCG60                TGGATGGCGTCTCCAGGCGATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGGTCGA120               GTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCA180               GTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAA240               AGTCGAGTTTACCACTCCCTACCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCC300               CTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAA360               AGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGCTCGG420               TACCCGGGTCGAGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGA480               ACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGG540               ACCGATCCAGCCTCCGCGGCCCCGAATTC569                                              (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 520 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AGATCTGCAGGGTCGCTCGGTGTTCGAGGCCACACGCGTCACCTTAATATGCGAAGTGGA60                CCGGATCTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACC120               ACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAG180               AGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAG240               TTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAG300               TGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAA360               GTCGAGCTCGGTACCCGGGTCGAGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGC420               TCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAG480               AAGACACCGGGACCGATCCAGCCTCCGCGGCCCCGAATTC520                                   (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 450 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Human cytomegalovirus                                           (B) STRAIN: K12, Towne                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: mRNA                                                            (B) LOCATION: 382..450                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GAATTCCTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACC60                ACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAG120               AGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAG180               TTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAG240               TGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAA300               GTCGAGCTCGGTACCCGGGTCGAGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGC360               TCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAG420               AAGACACCGGGACCGATCCAGCCTCCGCGG450                                             (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 450 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Human cytomegalovirus                                           (B) STRAIN: Towne                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: mRNA                                                            (B) LOCATION: 382..450                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GAATTCCTCGACCCGGGTACCGAGCTCGACTTTCACTTTTCTCTATCACTGATAGGGAGT60                GGTAAACTCGACTTTCACTTTTCTCTATCACTGATAGGGAGTGGTAAACTCGACTTTCAC120               TTTTCTCTATCACTGATAGGGAGTGGTAAACTCGACTTTCACTTTTCTCTATCACTGATA180               GGGAGTGGTAAACTCGACTTTCACTTTTCTCTATCACTGATAGGGAGTGGTAAACTCGAC240               TTTCACTTTTCTCTATCACTGATAGGGAGTGGTAAACTCGACTTTCACTTTTCTCTATCA300               CTGATAGGGAGTGGTAAACTCGAGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGC360               TCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAG420               AAGACACCGGGACCGATCCAGCCTCCGCGG450                                             (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 398 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Herpes Simplex Virus                                            (B) STRAIN: KOS                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GAGCTCGACTTTCACTTTTCTCTATCACTGATAGGGAGTGGTAAACTCGACTTTCACTTT60                TCTCTATCACTGATAGGGAGTGGTAAACTCGACTTTCACTTTTCTCTATCACTGATAGGG120               AGTGGTAAACTCGACTTTCACTTTTCTCTATCACTGATAGGGAGTGGTAAACTCGACTTT180               CACTTTTCTCTATCACTGATAGGGAGTGGTAAACTCGACTTTCACTTTTCTCTATCACTG240               ATAGGGAGTGGTAAACTCGACTTTCACTTTTCTCTATCACTGATAGGGAGTGGTAAACTC300               GAGATCCGGCGAATTCGAACACGCAGATGCAGTCGGGGCGGCGCGGTCCGAGGTCCACTT360               CGCATATTAAGGTGACGCGTGTGGCCTCGAACACCGAG398                                     (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      ACTTTATCACTGATAAACAAACTTATCAGTGATAAAGA38                                      (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      ACTCTATCATTGATAGAGTTCCCTATCAGTGATAGAGA38                                      (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      AGCTTATCATCGATAAGCTAGTTTATCACAGTTAAATT38                                      (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      ACTCTATCATTGATAGGGAACTCTATCAATGATAGGGA38                                      (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      AATCTATCACTGATAGAGTACCCTATCATCGATAGAGA38                                      (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 621 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTG48                            MetSerArgLeuAspLysSerLysValIleAsnSerAlaLeuGluLeu                              151015                                                                        CTTAATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAG96                            LeuAsnGluValGlyIleGluGlyLeuThrThrArgLysLeuAlaGln                              202530                                                                        AAGCTAGGTGTAGAGCAGCCTACATTGTATTGGCATGTAAAAAATAAG144                           LysLeuGlyValGluGlnProThrLeuTyrTrpHisValLysAsnLys                              354045                                                                        CGGGCTTTGCTCGACGCCTTAGCCATTGAGATGTTAGATAGGCACCAT192                           ArgAlaLeuLeuAspAlaLeuAlaIleGluMetLeuAspArgHisHis                              505560                                                                        ACTCACTTTTGCCCTTTAGAAGGGGAAAGCTGGCAAGATTTTTTACGT240                           ThrHisPheCysProLeuGluGlyGluSerTrpGlnAspPheLeuArg                              65707580                                                                      AATAAGGCTAAAAGTTTTAGATGTGCTTTACTAAGTCATCGCGATGGA288                           AsnLysAlaLysSerPheArgCysAlaLeuLeuSerHisArgAspGly                              859095                                                                        GCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACT336                           AlaLysValHisLeuGlyThrArgProThrGluLysGlnTyrGluThr                              100105110                                                                     CTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAG384                           LeuGluAsnGlnLeuAlaPheLeuCysGlnGlnGlyPheSerLeuGlu                              115120125                                                                     AATGCATTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGC432                           AsnAlaLeuTyrAlaLeuSerAlaValGlyHisPheThrLeuGlyCys                              130135140                                                                     GTATTGGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACA480                           ValLeuGluAspGlnGluHisGlnValAlaLysGluGluArgGluThr                              145150155160                                                                  CCTACTACTGATAGTATGCCGCCATTATTACGACAAGCTATCGAATTA528                           ProThrThrAspSerMetProProLeuLeuArgGlnAlaIleGluLeu                              165170175                                                                     TTTGATCACCAAGGTGCAGAGCCAGCCTTCTTATTCGGCCTTGAATTG576                           PheAspHisGlnGlyAlaGluProAlaPheLeuPheGlyLeuGluLeu                              180185190                                                                     ATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTGGGTCC621                              IleIleCysGlyLeuGluLysGlnLeuLysCysGluSerGlySer                                 195200205                                                                     (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 207 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      MetSerArgLeuAspLysSerLysValIleAsnSerAlaLeuGluLeu                              151015                                                                        LeuAsnGluValGlyIleGluGlyLeuThrThrArgLysLeuAlaGln                              202530                                                                        LysLeuGlyValGluGlnProThrLeuTyrTrpHisValLysAsnLys                              354045                                                                        ArgAlaLeuLeuAspAlaLeuAlaIleGluMetLeuAspArgHisHis                              505560                                                                        ThrHisPheCysProLeuGluGlyGluSerTrpGlnAspPheLeuArg                              65707580                                                                      AsnLysAlaLysSerPheArgCysAlaLeuLeuSerHisArgAspGly                              859095                                                                        AlaLysValHisLeuGlyThrArgProThrGluLysGlnTyrGluThr                              100105110                                                                     LeuGluAsnGlnLeuAlaPheLeuCysGlnGlnGlyPheSerLeuGlu                              115120125                                                                     AsnAlaLeuTyrAlaLeuSerAlaValGlyHisPheThrLeuGlyCys                              130135140                                                                     ValLeuGluAspGlnGluHisGlnValAlaLysGluGluArgGluThr                              145150155160                                                                  ProThrThrAspSerMetProProLeuLeuArgGlnAlaIleGluLeu                              165170175                                                                     PheAspHisGlnGlyAlaGluProAlaPheLeuPheGlyLeuGluLeu                              180185190                                                                     IleIleCysGlyLeuGluLysGlnLeuLysCysGluSerGlySer                                 195200205                                                                     (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 621 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTG48                            MetSerArgLeuAspLysSerLysValIleAsnSerAlaLeuGluLeu                              151015                                                                        CTTAATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAG96                            LeuAsnGluValGlyIleGluGlyLeuThrThrArgLysLeuAlaGln                              202530                                                                        AAGCTAGGTGTAGAGCAGCCTACACTGTATTGGCATGTAAAAAATAAG144                           LysLeuGlyValGluGlnProThrLeuTyrTrpHisValLysAsnLys                              354045                                                                        CGGGCTTTGCTCGACGCCTTAGCCATTGAGATGTTAGATAGGCACCAT192                           ArgAlaLeuLeuAspAlaLeuAlaIleGluMetLeuAspArgHisHis                              505560                                                                        ACTCACTTTTGCCCTTTAAAAGGGGAAAGCTGGCAAGATTTTTTACGC240                           ThrHisPheCysProLeuLysGlyGluSerTrpGlnAspPheLeuArg                              65707580                                                                      AATAAGGCTAAAAGTTTTAGATGTGCTTTACTAAGTCATCGCAATGGA288                           AsnLysAlaLysSerPheArgCysAlaLeuLeuSerHisArgAsnGly                              859095                                                                        GCAAAAGTACATTCAGATACACGGCCTACAGAAAAACAGTATGAAACT336                           AlaLysValHisSerAspThrArgProThrGluLysGlnTyrGluThr                              100105110                                                                     CTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAG384                           LeuGluAsnGlnLeuAlaPheLeuCysGlnGlnGlyPheSerLeuGlu                              115120125                                                                     AATGCATTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGC432                           AsnAlaLeuTyrAlaLeuSerAlaValGlyHisPheThrLeuGlyCys                              130135140                                                                     GTATTGGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACA480                           ValLeuGluAspGlnGluHisGlnValAlaLysGluGluArgGluThr                              145150155160                                                                  CCTACTACTGATAGTATGCCGCCATTATTACGACAAGCTATCGAATTA528                           ProThrThrAspSerMetProProLeuLeuArgGlnAlaIleGluLeu                              165170175                                                                     TTTGATCACCAAGGTGCAGAGCCAGCCTTCTTATTCGGCCTTGAATTG576                           PheAspHisGlnGlyAlaGluProAlaPheLeuPheGlyLeuGluLeu                              180185190                                                                     ATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTGGGTCC621                              IleIleCysGlyLeuGluLysGlnLeuLysCysGluSerGlySer                                 195200205                                                                     (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 207 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      MetSerArgLeuAspLysSerLysValIleAsnSerAlaLeuGluLeu                              151015                                                                        LeuAsnGluValGlyIleGluGlyLeuThrThrArgLysLeuAlaGln                              202530                                                                        LysLeuGlyValGluGlnProThrLeuTyrTrpHisValLysAsnLys                              354045                                                                        ArgAlaLeuLeuAspAlaLeuAlaIleGluMetLeuAspArgHisHis                              505560                                                                        ThrHisPheCysProLeuLysGlyGluSerTrpGlnAspPheLeuArg                              65707580                                                                      AsnLysAlaLysSerPheArgCysAlaLeuLeuSerHisArgAsnGly                              859095                                                                        AlaLysValHisSerAspThrArgProThrGluLysGlnTyrGluThr                              100105110                                                                     LeuGluAsnGlnLeuAlaPheLeuCysGlnGlnGlyPheSerLeuGlu                              115120125                                                                     AsnAlaLeuTyrAlaLeuSerAlaValGlyHisPheThrLeuGlyCys                              130135140                                                                     ValLeuGluAspGlnGluHisGlnValAlaLysGluGluArgGluThr                              145150155160                                                                  ProThrThrAspSerMetProProLeuLeuArgGlnAlaIleGluLeu                              165170175                                                                     PheAspHisGlnGlyAlaGluProAlaPheLeuPheGlyLeuGluLeu                              180185190                                                                     IleIleCysGlyLeuGluLysGlnLeuLysCysGluSerGlySer                                 195200205                                                                     (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 192 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      GACATGGAAAAAGCGACACCGGAGACGATGGTCCATTGGATTTGTCTG48                            AspMetGluLysAlaThrProGluThrMetValHisTrpIleCysLeu                              151015                                                                        AAGATGGAGCCAGCTCTGTGGATGGCCATTACAGCAACATCGCACGGC96                            LysMetGluProAlaLeuTrpMetAlaIleThrAlaThrSerHisGly                              202530                                                                        GCAAGGCACAGGACATTCGTCGGGTTTTCCGGCTGCCTCCACCGCAAA144                           AlaArgHisArgThrPheValGlyPheSerGlyCysLeuHisArgLys                              354045                                                                        TCCCTCACGTACCCAGTGATATGCCTGAGCAAACCGAGCCAGAGGATT192                           SerLeuThrTyrProValIleCysLeuSerLysProSerGlnArgIle                              505560                                                                        (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 64 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      AspMetGluLysAlaThrProGluThrMetValHisTrpIleCysLeu                              151015                                                                        LysMetGluProAlaLeuTrpMetAlaIleThrAlaThrSerHisGly                              202530                                                                        AlaArgHisArgThrPheValGlyPheSerGlyCysLeuHisArgLys                              354045                                                                        SerLeuThrTyrProValIleCysLeuSerLysProSerGlnArgIle                              505560                                                                        (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 816 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      CTGGACGACTCGAAGCGCGTAGCCAAGCGGAAGCTGATCGAGGAGAAC48                            LeuAspAspSerLysArgValAlaLysArgLysLeuIleGluGluAsn                              151015                                                                        CGGGAGCGGCGACGCAAGGAGGAGATGATCAAATCCCTGCAGCACCGG96                            ArgGluArgArgArgLysGluGluMetIleLysSerLeuGlnHisArg                              202530                                                                        CCCAGCCCCAGCGCAGAGGAGTGGGAGCTGATCCACGTGGTGACCGAG144                           ProSerProSerAlaGluGluTrpGluLeuIleHisValValThrGlu                              354045                                                                        GCGCACCGCAGCACCAACGCGCAGGGCAGCCACTGGAAGCAGAGGAGG192                           AlaHisArgSerThrAsnAlaGlnGlySerHisTrpLysGlnArgArg                              505560                                                                        AAATTCCTGCTCGAAGATATCGGTCAGTCGCCCATGGCCTCCATGCTT240                           LysPheLeuLeuGluAspIleGlyGlnSerProMetAlaSerMetLeu                              65707580                                                                      GACGGGGACAAAGTGGACCTGGAGGCGTTCAGCGAGTTTACAAAAATC288                           AspGlyAspLysValAspLeuGluAlaPheSerGluPheThrLysIle                              859095                                                                        ATCACGCCGGCCATCACCCGCGTGGTCGACTTTGCCAAAAACCTGCCC336                           IleThrProAlaIleThrArgValValAspPheAlaLysAsnLeuPro                              100105110                                                                     ATGTTCTCGGAGCTGCCGTGCGAGGATCAGATCATCCTGCTGAAGGGC384                           MetPheSerGluLeuProCysGluAspGlnIleIleLeuLeuLysGly                              115120125                                                                     TGCTGCATGGAGATCATGTCGCTGCGCGCCGCCGTGCGCTACGACCCC432                           CysCysMetGluIleMetSerLeuArgAlaAlaValArgTyrAspPro                              130135140                                                                     GAGAGCGAAACGCTGACGCTGAGCGGGGAAATGGCCGTCAAACGCGAG480                           GluSerGluThrLeuThrLeuSerGlyGluMetAlaValLysArgGlu                              145150155160                                                                  CAGTTGAAGAACGGAGGGCTGGGGGTCGTGTCTGATGCCATCTTCGAC528                           GlnLeuLysAsnGlyGlyLeuGlyValValSerAspAlaIlePheAsp                              165170175                                                                     CTCGGCAAGTCGCTGTCTGCCTTCAACCTGGACGACACCGAGGTGGCC576                           LeuGlyLysSerLeuSerAlaPheAsnLeuAspAspThrGluValAla                              180185190                                                                     CTGCTGCAGGCCGTGCTGCTCATGTCCTCAGACCGGACGGGGCTGATC624                           LeuLeuGlnAlaValLeuLeuMetSerSerAspArgThrGlyLeuIle                              195200205                                                                     TGCGTGGATAAGATAGAGAAGTGCCAGGAGTCGTACCTGCTGGCGTTC672                           CysValAspLysIleGluLysCysGlnGluSerTyrLeuLeuAlaPhe                              210215220                                                                     GAGCACTACATCAACTACCGCAAACACAACATTCCCCACTTCTGGTCC720                           GluHisTyrIleAsnTyrArgLysHisAsnIleProHisPheTrpSer                              225230235240                                                                  AAGCTGCTGATGAAGGTGGCGGACCTGCGCATGATCGGCGCCTACCAC768                           LysLeuLeuMetLysValAlaAspLeuArgMetIleGlyAlaTyrHis                              245250255                                                                     GCCAGCCGCTTCCTGCACATGAAGGTGGAGTGCCCCACCGAGCTCTCC816                           AlaSerArgPheLeuHisMetLysValGluCysProThrGluLeuSer                              260265270                                                                     (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 272 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      LeuAspAspSerLysArgValAlaLysArgLysLeuIleGluGluAsn                              151015                                                                        ArgGluArgArgArgLysGluGluMetIleLysSerLeuGlnHisArg                              202530                                                                        ProSerProSerAlaGluGluTrpGluLeuIleHisValValThrGlu                              354045                                                                        AlaHisArgSerThrAsnAlaGlnGlySerHisTrpLysGlnArgArg                              505560                                                                        LysPheLeuLeuGluAspIleGlyGlnSerProMetAlaSerMetLeu                              65707580                                                                      AspGlyAspLysValAspLeuGluAlaPheSerGluPheThrLysIle                              859095                                                                        IleThrProAlaIleThrArgValValAspPheAlaLysAsnLeuPro                              100105110                                                                     MetPheSerGluLeuProCysGluAspGlnIleIleLeuLeuLysGly                              115120125                                                                     CysCysMetGluIleMetSerLeuArgAlaAlaValArgTyrAspPro                              130135140                                                                     GluSerGluThrLeuThrLeuSerGlyGluMetAlaValLysArgGlu                              145150155160                                                                  GlnLeuLysAsnGlyGlyLeuGlyValValSerAspAlaIlePheAsp                              165170175                                                                     LeuGlyLysSerLeuSerAlaPheAsnLeuAspAspThrGluValAla                              180185190                                                                     LeuLeuGlnAlaValLeuLeuMetSerSerAspArgThrGlyLeuIle                              195200205                                                                     CysValAspLysIleGluLysCysGlnGluSerTyrLeuLeuAlaPhe                              210215220                                                                     GluHisTyrIleAsnTyrArgLysHisAsnIleProHisPheTrpSer                              225230235240                                                                  LysLeuLeuMetLysValAlaAspLeuArgMetIleGlyAlaTyrHis                              245250255                                                                     AlaSerArgPheLeuHisMetLysValGluCysProThrGluLeuSer                              260265270                                                                     (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      TCCCCGGGTAACTAAGTAAGGATCC25                                                   (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      AGTGGGTCCCCGGGTGACATGGAA24                                                    (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: polypeptide                                               (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      SerGlySerProGlyAspMetGlu                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      AGTGGGTCCCCGGGTCTGGACGAC24                                                    (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: polypeptide                                               (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      SerGlySerProGlyLeuAspAsp                                                      15                                                                            __________________________________________________________________________

We claim:
 1. An isolated nucleic acid encoding a fusion protein whichinhibits transcription in eukaryotic cells, the fusion proteincomprising a first polypeptide which binds to tet operator sequences,operatively linked to a heterologous second polypeptide which inhibitstranscription in eukaryotic cells.
 2. The nucleic acid of claim 1,wherein the first polypeptide binds to tet operator sequences in theabsence but not the presence of tetracycline or a tetracycline analogue.3. The nucleic acid of claim 2, wherein the first polypeptide is a Tetrepressor.
 4. The nucleic acid of claim 3, wherein the first polypeptidecomprises an amino acid sequence shown in SEQ ID NO:
 17. 5. The nucleicacid of claim 1, wherein the first polypeptide binds to tet operatorsequences in the presence but not the absence of tetracycline or atetracycline analogue.
 6. The nucleic acid of claim 5, wherein the firstpolypeptide is a mutated Tet repressor.
 7. The nucleic acid of claim 6,wherein the mutated Tet repressor has at least one amino acidsubstitution compared to a wild-type Tet repressor.
 8. The nucleic acidof claim 6, wherein the mutated Tet repressor has at least one aminoacid addition or deletion compared to a wild-type Tet repressor.
 9. Thenucleic acid of claim 7, wherein the mutated Tet repressor has an aminoacid substitution at at least one amino acid position corresponding toan amino acid position selected from the group consisting of position71, position 95, position 101 and position 102 of a wild-typeTn10-derived Tet repressor amino acid sequence.
 10. The nucleic acid ofclaim 9, wherein the mutated Tet repressor comprises an amino acidsequence shown in SEQ ID NO:
 19. 11. The nucleic acid of claim 1,wherein the second polypeptide comprises a transcription silencer domainof a v-erbA oncogene product.
 12. The nucleic acid of claim 11, whereinthe second polypeptide comprises an amino acid sequence shown in SEQ IDNO:
 23. 13. The nucleic acid of claim 1, wherein the second polypeptidecomprises a transcription silencer domain of a Drosophila Krueppelprotein.
 14. The nucleic acid of claim 13, wherein the secondpolypeptide comprises an amino acid sequence shown in SEQ ID NO:
 21. 15.The nucleic acid of claim 1, wherein the second polypeptide comprises atranscription silencer domain of a protein selected from the groupconsisting of the retinoic acid receptor alpha, the thyroid hormonereceptor alpha, the yeast Ssn6/Tup1 protein complex, the Drosophilaprotein even-skipped, SIR1, NeP1, the Drosophila dorsal protein, TSF3,SFI, the Drosophila hunchback protein, the Drosophila knirps protein,WT1, Oct-2.1, the Drosophila engrailed protein, E4BP4 and ZF5.
 16. Thenucleic acid molecule of claim 1, wherein the fusion protein furthercomprises an operatively linked third polypeptide which promotestransport of the fusion protein to a cell nucleus.
 17. A recombinantvector comprising the nucleic acid molecule of claim 2 in a formsuitable for expression of the fusion protein in a host cell.
 18. A hostcell comprising the recombinant vector of claim
 17. 19. The host cell ofclaim 18, further comprising a nucleotide sequence to be transcribedoperatively linked to at least one tet operator sequence.
 20. The hostcell of claim 19, wherein the nucleotide sequence to be transcribed isan exogenous nucleotide sequence introduced into the host cell.
 21. Thehost cell of claim 19, wherein the nucleotide sequence to be transcribedis an endogenous nucleotide sequence to which at least one tet operatorsequence has been operatively linked.
 22. The host cell of claim 19,further comprising nucleic acid encoding a fusion protein whichactivates transcription in eukaryotic cells, the fusion proteincomprising a first polypeptide which binds to a tet operator sequenceeither: (i) in the absence but not the presence of tetracycline or atetracycline analogue or (ii) in the presence but not the absence oftetracycline or a tetracycline analogue, operatively linked to aheterologous second polypeptide which activates transcription ineukaryotic cells.
 23. The host cell of claim 19, which is a mammaliancell.
 24. The host cell of claim 23, which is a human cell.
 25. The hostcell of claim 19, which is a yeast, insect or fungal cell.
 26. A methodfor inhibiting transcription of a nucleotide sequence operatively linkedto the at least one tet operator sequence in the host cell of claim 19in vitro, comprising decreasing the concentration of tetracycline or atetracycline analogue that is in contact with the host cell in vitro.27. A recombinant vector comprising the nucleic acid molecule of claim 5in a form suitable for expression of the fusion protein in a host cell.28. A host cell comprising the recombinant vector of claim
 27. 29. Thehost cell of claim 28, further comprising a nucleotide sequence to betranscribed operatively linked to at least one tet operator sequence.30. The host cell of clam 29, wherein the nucleotide sequence to betranscribed is an exogenous nucleotide sequence introduced into the hostcell.
 31. The host cell of claim 29, wherein the nucleotide sequence tobe transcribed is an endogenous nucleotide sequence to which at leastone tet operator sequence has been operatively linked.
 32. The host cellof claim 29, further comprising nucleic acid encoding a fusion proteinwhich activates transcription in eukaryotic cells, the fusion proteincomprising a first polypeptide which binds to a tet operator sequenceeither (i) in the absence but not the presence of tetracycline or atetracycline analogue or (ii) in the presence but not the absence oftetracycline or a tetracycline analogue, operatively linked to aheterologous second polypeptide which activates transcription ineukaryotic cells.
 33. The host cell of claim 29, which is a mammaliancell.
 34. The host cell of claim 33, which is a human cell.
 35. The hostcell of claim 29, which is a yeast, insect or fungal cell.
 36. A methodfor inhibiting transcription of the nucleotide sequence operativelylinked to the at least one tet operator sequence in the host cell ofclaim 29 in vitro, comprising increasing the concentration oftetracycline or a tetracycline analogue that is in contact with the hostcell in vitro.
 37. A recombinant vector suitable for homologousrecombination comprising the nucleic acid of claim 1, wherein thenucleic acid encoding the fusion protein is flanked at its 5' and 3'ends by additional nucleic acid of a eukaryotic gene, the additionalnucleic acid being of sufficient length for successful homologousrecombination with the eukaryotic gene.
 38. The recombinant vector ofclaim 17, wherein expression of the fusion protein is regulated by atleast one tet operator sequence.
 39. The recombinant vector of claim 17,wherein expression of the fusion protein is regulated by at least onevirally-derived regulatory element.
 40. The recombinant vector of claim17, wherein expression of the fusion protein is regulated by at leastone tissue specific regulatory element.
 41. The host cell of claim 19,which is a plant.
 42. The recombinant vector of claim 27, whereinexpression of the fusion protein is regulated by at least one tetoperator sequence.
 43. The recombinant vector of claim 27, whereinexpression of the fusion protein is regulated by at least onevirally-derived regulatory element.
 44. The recombinant vector of claim27, wherein expression of the fusion protein is regulated by at leastone tissue specific regulatory element.
 45. The host cell of claim 29,which is a plant.
 46. A kit comprising a carrier means having in closeconfinement therein at least two container means comprising:a) a firstcontainer means containing a first nucleic acid encoding a fusionprotein which inhibits transcription in eukaryotic cells, the fusionprotein comprising a first polypeptide which binds to tet operatorsequences either (I) in the presence but not the absence of tetracyclineor a tetracycline analogue or (ii) in the absence but not the presenceof tetracycline or a tetracycline analogue, operatively linked to aheterologous second polypeptide which inhibits transcription ineukaryotic cells, or a eukaryotic cell line into which said firstnucleic acid has been stably introduced; and b) a second container meanscontaining a second nucleic acid comprising a cloning site forintroduction of a nucleotide sequence to be transcribed operativelylinked to at least one tet operator sequence.
 47. The kit of claim 46,further comprising:c) a third container means containing a third nucleicacid encoding a fusion protein which activates transcription ineukaryotic cells, the fusion protein comprising a first polypeptidewhich binds to tet operator sequences either (i) in the presence but notthe absence of tetracycline or a tetracycline analogue or (ii) in theabsence but not the presence of tetracycline or a tetracycline analogue,operatively linked to a heterologous second polypeptide which activatestranscription in eukaryotic cells.
 48. The kit of claim 46, wherein theeukaryotic cell line into which the first nucleic acid has beenintroduced further contains nucleic acid encoding a fusion protein whichactivates transcription in eukaryotic cells, the fusion proteincomprising a first polypeptide which binds to tet operator sequenceseither (i) in the presence but not the absence of tetracycline or atetracycline analogue or (ii) in the absence but not the presence oftetracycline or a tetracycline analogue, operatively linked to aheterologous second polypeptide which activates transcription ineukaryotic cells.
 49. The kit of claim 46, further comprising a thirdcontainer means containing a tetracycline or a tetracycline analogue.